Guided microbial remodeling, a platform for the rational improvement of microbial species for agriculture

ABSTRACT

The present disclosure provides guided microbial remodeling (GMR) methods for the rational improvement of plant-associated microbes to perform plant-beneficial functions. The GMR methods described herein allow for non-intergeneric genetic optimization of key regulatory networks within the microbes, which improve plant-beneficial functions over wild-type microbes but don&#39;t have the risks associated with transgenic approaches (e.g., unpredictable gene function, public and regulatory concerns, etc.). The present disclosure also provides remodeled microbes and compositions thereof. The utilization of remodeled microbes and compositions thereof will enable farmers to realize more productive and predictable crop yields without the nutrient degradation, leaching, or toxic runoff associated with traditional synthetically derived fertilizers.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of International PCTApplication No. PCT/US2019/039528, filed Jun. 27, 2019, which claims thebenefit of priority to U.S. Provisional Application No. 62/690,619,filed on Jun. 27, 2018, which is incorporated by reference herein in itsentirety.

INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing filename:PIVO_007_01WO_SeqList_ST25.txt, date created, Jun. 26, 2019, file size602 kilobytes.

BACKGROUND OF THE DISCLOSURE

By 2050 the United Nations' Food and Agriculture Organization projectsthat total food production must increase by 70% to meet the needs of agrowing population, a challenge that is exacerbated by numerous factors,including: diminishing freshwater resources, increasing competition forarable land, rising energy prices, increasing input costs, and thelikely need for crops to adapt to the pressures of a drier, hotter, andmore extreme global climate.

Current agricultural practices are not well equipped to meet thisgrowing demand for food production, while simultaneously balancing theenvironmental impacts that result from increased agricultural intensity.

One of the major agricultural inputs needed to satisfy global fooddemand is nitrogen fertilizer. However, the current industrial standardutilized to produce nitrogen fertilizer, is an artificial nitrogenfixation method called the Haber-Bosch process, which convertsatmospheric nitrogen (N₂) to ammonia (NH₃) by a reaction with hydrogen(H₂) using a metal catalyst under high temperatures and pressures. Thisprocess is resource intensive and deleterious to the environment.

In contrast to the synthetic Haber-Bosch process, certain biologicalsystems have evolved to fix atmospheric nitrogen. These systems utilizean enzyme called nitrogenase that catalyzes the reaction between N₂ andH₂, and results in nitrogen fixation. For example, rhizobia arediazotrophic bacteria that fix nitrogen after becoming establishedinside root nodules of legumes. An important goal of nitrogen fixationresearch is the extension of this phenotype to non-leguminous plants,particularly to important agronomic grasses such as wheat, rice, andcorn. However, despite the significant progress made in understandingthe development of the nitrogen-fixing symbiosis between rhizobia andlegumes, the path to use that knowledge to induce nitrogen-fixingnodules on non-leguminous crops is still not clear.

Consequently, the vast majority of modern row crop agriculture utilizesnitrogen fertilizer that is produced via the resource intensive andenvironmentally deleterious Haber-Bosch process. For instance, the USDAindicates that the average U.S. corn farmer typically applies between130 and 200 lb. of nitrogen per acre (146 to 224 kg/ha). This nitrogenis not only produced in a resource intensive synthetic process, but isapplied by heavy machinery crossing/impacting the field's soil, burningpetroleum, and requiring hours of human labor.

Furthermore, the nitrogen fertilizer produced by the industrialHaber-Bosch process is not well utilized by the target crop. Rain,runoff, heat, volatilization, and the soil microbiome degrade theapplied chemical fertilizer. This equates to not only wasted money, butalso adds to increased pollution instead of harvested yield. To thisend, the United Nations has calculated that nearly 80% of fertilizer islost before a crop can utilize it. Consequently, modern agriculturalfertilizer production and delivery is not only deleterious to theenvironment, but it is extremely inefficient.

In order to meet the world's growing food supply needs—while alsobalancing resource utilization and providing minimal impacts uponenvironmental systems—a better approach to nitrogen fixation anddelivery to plants is urgently needed.

SUMMARY OF THE DISCLOSURE

Provided herein are guided microbial remodeling (GMR) methods for therational improvement of plant-associated microbes to performplant-beneficial functions.

In one aspect, the GMR method comprises: (a) providing a plurality ofmicrobial species that are associated with a target plant of interest;(b) assaying the plurality of microbial species for: colonizationmetrics, and ability to perform a plant-beneficial function of interest;(c) selecting a candidate microbial species from the plurality ofassayed microbial species; (d) introducing one or more targetednon-intergeneric genetic variations into the candidate microbialspecies; (e) confirming integration of the non-intergeneric geneticvariation at the target genomic locus and absence of any transgeneticsequence; and (f) repeating steps d) and e) one or more times, until thecandidate microbial species has acquired an improved ability to performthe plant-beneficial function of interest.

In one aspect, the step b) of assaying the plurality of microbialspecies comprises assaying the plurality of microbial species fortranscriptionally active genes under metabolically relevantenvironmental conditions.

In one aspect, the GMR method comprises a step of sequencing the genomesof the plurality of microbial species obtained in step a) andcharacterizing one or more genomic pathways, or sets of genes, that areassociated with a plant-beneficial function of interest.

In one aspect, the step d) of introducing one or more targetednon-intergeneric genetic variations into the candidate microbial speciescomprises: (a) transforming the candidate microbial species with atransformation plasmid comprising (i) a selection marker, (ii) acounterselection marker, (iii) a DNA fragment comprising: anon-intergeneric genetic variation to be introduced into the candidatemicrobial species at a target genomic locus in one or more genomicpathways, or sets of genes, that are associated with a plant-beneficialfunction of interest, and homology arms to the target genomic locusflanking the non-intergeneric genetic variation, and (iv) plasmidbackbone; (b) selecting for a candidate microbial species that hasundergone an initial homologous recombination and has thenon-intergeneric genetic variation integrated into the target genomiclocus based on the presence of the selection marker in the genome; and(c) selecting for a candidate microbial species that has thenon-intergeneric genetic variation integrated into the target genomiclocus, but has undergone an additional homologous recombination thatloops-out the plasmid backbone, based on the absence of thecounterselection marker.

In one aspect, the step e) of confirming integration of thenon-intergeneric genetic variation at the target genomic locus andabsence of any transgenetic sequence comprises sequencing the genome ofthe transformed candidate microbial species and confirming absence ofany transgenetic sequence from the genome of the transformed candidatemicrobial species. In one aspect, the step e) comprises confirmingabsence of any transgenetic sequence from the transformation plasmid.

In one aspect, the GMR method comprises: (a) providing a plurality ofmicrobial species that are associated with a target plant of interest;(b) sequencing the genomes of the plurality of microbial species andcharacterizing one or more genomic pathways, or sets of genes, that areassociated with a plant-beneficial function of interest; (c) assayingthe plurality of microbial species for: colonization metrics,transcriptionally active genes under metabolically relevantenvironmental conditions, and ability to perform a plant-beneficialfunction of interest; (d) selecting a candidate microbial species fromthe plurality of assayed microbial species; (e) transforming thecandidate microbial species with a transformation plasmid comprising:(i) a selection marker, (ii) a counterselection marker, (iii) a DNAfragment comprising: a non-intergeneric genetic variation to beintroduced into the candidate microbial species at a target genomiclocus in one or more genomic pathways, or sets of genes, that areassociated with a plant-beneficial function of interest, and homologyarms to the target genomic locus flanking the non-intergeneric geneticvariation; and (iv) plasmid backbone; (f) selecting for a candidatemicrobial species that has undergone an initial homologous recombinationand has the non-intergeneric genetic variation integrated into thetarget genomic locus based on the presence of the selection marker inthe genome; (g) selecting for a candidate microbial species that has thenon-intergeneric genetic variation integrated into the target genomiclocus, but has undergone an additional homologous recombination thatloops-out the plasmid backbone, based on the absence of thecounterselection marker; (h) sequencing the genome of the transformedcandidate microbial species and confirming integration of thenon-intergeneric genetic variation at the target genomic locus andabsence of any genetic sequence from the transformation plasmid; and (i)repeating steps e)-h) one or more times, until a candidate microbialspecies has acquired an improved ability to perform the plant-beneficialfunction of interest.

In one aspect, the plant-beneficial functions of interest in the methodsdescribed herein can be nitrogen fixation, phosphate solubilization,microbial colonization, or combinations thereof.

In one aspect, the step of sequencing the genomes of the plurality ofmicrobial species and characterizing one or more genomic pathways, orsets of genes, that are associated with a plant-beneficial function ofinterest (e.g. step b of the method described above) comprises wholegenome sequencing and/or characterizing the nitrogen fixation pathway.

In one aspect, characterizing the nitrogen fixation pathway comprisescharacterizing a set of genes selected from the group consisting of:nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase,glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD,glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW,nifZ, nifM, nifF, nifB, nifQ, a gene associated with biosynthesis of anitrogenase enzyme, and combinations thereof.

In one aspect, characterizing the microbial colonization pathwaycomprises characterizing one or more genes involved in a pathwayselected from the group consisting of: exopolysaccharide production,endo-polygalaturonase production, trehalose production, and glutamineconversion.

In one aspect, characterizing the microbial colonization pathwaycomprises characterizing one or more genes selected from the groupconsisting of: bcsii, bcsiii, yjbE, fhaB, pehA, otsB, treZ, glsA2, andcombinations thereof.

In one aspect, characterizing the microbial colonization pathwaycomprises characterizing one or more genes selected from the groupconsisting of: nifA, nijL, ntrB, ntrC, polynucleotide encoding glutaminesynthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encodingglutaminase, glnD, glnE, nifJ, nifH, nifD, nijK, nifY, nijE, nijN, nifU,nijS, nifV, nifW, nijZ, nifM, nijF, nijB, nifQ, a gene associated withbiosynthesis of a nitrogenase enzyme, bcsii, bcsiii, yjbE, fhaB, pehA,otsB, treZ, glsA2, and combinations thereof.

In one aspect, the step of assaying the plurality of microbial speciesfor colonization metrics (e.g. in step c of the method described above)comprises assaying the plurality of microbial species for colonizationmetrics under greenhouse or lab based conditions and/or under fieldconditions.

In one aspect, the colonization metric comprises at least one of thefollowing: spatial colonization patterns, temporal colonizationdynamics, density of colonization, or combinations thereof.

In one aspect, the step of assaying the plurality of microbial speciesfor transcriptionally active genes under metabolically relevantenvironmental conditions (e.g. in step c of the method described above)occurs under greenhouse or lab based conditions.

In one aspect, the step of assaying the plurality of microbial speciesfor transcriptionally active genes under metabolically relevantenvironmental conditions (e.g. in step c of the method described above)occurs under field conditions.

In one aspect, the step of assaying the plurality of microbial speciesfor transcriptionally active genes under metabolically relevantenvironmental conditions (e.g. in step c of the method described above)occurs in vitro.

In one aspect, the step of assaying the plurality of microbial speciesfor transcriptionally active genes under metabolically relevantenvironmental conditions (e.g. in step c of the method described above)occurs under greenhouse or lab based conditions; under field conditions;and/or in vitro.

In one aspect, the step of assaying the plurality of microbial speciesfor transcriptionally active genes under metabolically relevantenvironmental conditions (e.g. in step c of the method described above)occurs under greenhouse or lab based conditions and comprises (i)measuring the transcriptomic profile of the microbial species; (ii)measuring the transcriptomic activity of genes associated with themicrobial species' ability to perform a plant-beneficial function ofinterest; (iii) measuring the transcriptomic activity of regulatory genesequences; (iv) measuring the transcriptomic activity of promotersequences; (v) measuring the transcriptomic activity of promotersequences in the presence of exogenous nitrogen; and/or (vi) measuringthe transcriptomic activity of promoter sequences in the presence ofexogenous nitrogen, wherein said transcriptomic activity of the promotersequences is measured by quantifying the expression of a regulated gene.

In one aspect, the step of assaying the plurality of microbial speciesfor transcriptionally active genes under metabolically relevantenvironmental conditions (e.g. in step c of the method described above)occurs under field conditions and comprises (i) measuring thetranscriptomic profile of the microbial species; (ii) measuring thetranscriptomic activity of genes associated with the microbial species'ability to perform a plant-beneficial function of interest; (iii)measuring the transcriptomic activity of regulatory gene sequences; (iv)measuring the transcriptomic activity of promoter sequences; (v)measuring the transcriptomic activity of promoter sequences in thepresence of exogenous nitrogen; and/or (vi) measuring the transcriptomicactivity of promoter sequences in the presence of exogenous nitrogen,wherein said transcriptomic activity of the promoter sequences ismeasured by quantifying the expression of a regulated gene.

In one aspect, the step of assaying the plurality of microbial speciesfor transcriptionally active genes under metabolically relevantenvironmental conditions (e.g. in step c of the method described above)occurs in vitro and comprises (i) measuring the transcriptomic profileof the microbial species; (ii) measuring the transcriptomic activity ofgenes associated with the microbial species' ability to perform aplant-beneficial function of interest; (iii) measuring thetranscriptomic activity of regulatory gene sequences; (iv) measuring thetranscriptomic activity of promoter sequences; (v) measuring thetranscriptomic activity of promoter sequences in nitrogen-depleted andnitrogen-replete conditions; and/or (vi) measuring the transcriptomicactivity of promoter sequences in nitrogen-depleted and nitrogen-repleteconditions, wherein said transcriptomic activity of the promotersequences is measured by quantifying the expression of a regulated gene.

In one aspect, the step of assaying the plurality of microbial speciesfor colonization metrics (e.g. in step c of the guided microbialremodeling method described above) comprises: growing said plurality ofmicrobial species in intimate association with a target plant.

In one aspect, the step of assaying the plurality of microbial speciesfor colonization metrics (e.g. in step c of the guided microbialremodeling method described above) comprises: growing said plurality ofmicrobial species in intimate association with a target plant undergreenhouse or lab based conditions.

In one aspect, the step of assaying the plurality of microbial speciesfor colonization metrics (e.g. in step c of the guided microbialremodeling method described above) comprises: growing said plurality ofmicrobial species in intimate association with a target plant underfield conditions.

In one aspect, the step of assaying the plurality of microbial speciesfor colonization metrics (e.g. in step c of the guided microbialremodeling method described above) comprises: growing said plurality ofmicrobial species in intimate association with a target plant undergreenhouse or lab based conditions and under field conditions.

In one aspect, the step of assaying the plurality of microbial speciesfor transcriptionally active genes under metabolically relevantenvironmental conditions (e.g. in step c of the guided microbialremodeling method described above) comprises: growing said plurality ofmicrobial species in intimate association with a target plant.

In one aspect, the step of assaying the plurality of microbial speciesfor transcriptionally active genes under metabolically relevantenvironmental conditions (e.g. in step c of the guided microbialremodeling method described above) comprises: growing said plurality ofmicrobial species in intimate association with a target plant undergreenhouse or lab based conditions.

In one aspect, the step of assaying the plurality of microbial speciesfor transcriptionally active genes under metabolically relevantenvironmental conditions (e.g. in step c of the guided microbialremodeling method described above) comprises: growing said plurality ofmicrobial species in intimate association with a target plant underfield conditions.

In one aspect, the step of assaying the plurality of microbial speciesfor transcriptionally active genes under metabolically relevantenvironmental conditions (e.g. in step c of the guided microbialremodeling method described above) comprises: growing said plurality ofmicrobial species in intimate association with a target plant undergreenhouse or lab based conditions and under field conditions.

In one aspect, the step of assaying the plurality of microbial speciesfor colonization metrics and transcriptionally active genes undermetabolically relevant environmental conditions (e.g. in step c of theguided microbial remodeling method described above) comprises: growingsaid plurality of microbial species in intimate association with atarget plant under field conditions.

In one aspect, the step of assaying the plurality of microbial speciesin step c of the guided microbial remodeling method described abovecomprises assaying the plurality of microbial species for ability toperform a plant-beneficial function of interest under greenhouse or labbased conditions.

In one aspect, the step of assaying the plurality of microbial speciesin step c of the guided microbial remodeling method described abovecomprises assaying the plurality of microbial species for nitrogenfixation activity. In one aspect, the nitrogen fixation activity isassayed in an acetylene reduction assay or ammonium excretion assay.

In one aspect, the transformation plasmid used in step e of the guidedmicrobial remodeling method described above is a suicide plasmid.

In one aspect, sequencing the genome of the transformed candidatemicrobial species and confirming integration of the non-intergenericgenetic variation at the target genomic locus and absence of any geneticsequence from the transformation plasmid (step h of the guided microbialremodeling method described above) comprises whole genome sequencing.

In one aspect, provided herein is a guided microbial remodeling methodfor the rational improvement of plant-associated microbes to performplant-beneficial functions, comprising: (a) providing a plurality ofmicrobial species; (b) assaying the plurality of microbial species for:colonization metrics, and ability to perform a plant-beneficial functionof interest; (c) selecting a candidate microbial species from theplurality of assayed microbial species; (d) introducing one or moretargeted non-intergeneric genetic variations into the candidatemicrobial species at a target genomic locus in one or more genomicpathways, or sets of genes, that are associated with a plant-beneficialfunction of interest; (e) confirming integration of the non-intergenericgenetic variation at the target genomic locus and absence of anytransgenic genetic sequence; and (0 repeating steps d)-e) one or moretimes, until a candidate microbial species has acquired an improvedability to perform the plant-beneficial function of interest. In afurther aspect of this embodiment, the step b) comprises assayingtranscriptionally active genes under metabolically relevantenvironmental conditions. In a further aspect of this embodiment, instep d), said non-intergeneric genetic variations are selected from thegroup consisting of: full gene deletions, partial gene deletions,promoter insertions, single base pair changes, and combinations thereof.In yet further aspect of this embodiment, step e) comprises sequencingthe genome of the candidate microbial species.

In one aspect, provided herein is A guided microbial remodeling methodfor the rational improvement of plant-associated microbes to performplant-beneficial functions, comprising: (a) providing a plurality ofmicrobial species; (b) assaying the plurality of microbial species for:colonization metrics, and ability to perform a plant-beneficial functionof interest; (c) selecting a candidate microbial species from theplurality of assayed microbial species; (d) introducing two or moretargeted non-intergeneric genetic variations into the candidatemicrobial species at two or more target genomic loci, in one or moregenomic pathways, or sets of genes, that are associated with aplant-beneficial function of interest; and (e) confirming introductionof the non-intergeneric genetic variations at the target genomic lociand absence of any transgenic genetic sequence. In a further aspect ofthis embodiment, step b) comprises assaying transcriptionally activegenes under metabolically relevant environmental conditions. In afurther aspect of this embodiment, in step d), said non-intergenericgenetic variations are selected from the group consisting of: full genedeletions, partial gene deletions, promoter insertions, single base pairchanges, and combinations thereof. In a further aspect of thisembodiment, step e) comprises sequencing the genome of the candidatemicrobial species.

In one aspect, provided herein is a guided microbial remodeling methodfor the rational improvement of plant-associated microbes to performplant-beneficial functions, comprising: (a) providing a plurality ofmicrobial species; (b) assaying the plurality of microbial species for:colonization metrics, transcriptionally active genes under metabolicallyrelevant environmental conditions, and ability to perform aplant-beneficial function of interest; (c) selecting a candidatemicrobial species from the plurality of assayed microbial species; (d)introducing one or more targeted non-intergeneric genetic variationsinto the candidate microbial species at a target genomic locus in one ormore genomic pathways, or sets of genes, that are associated with aplant-beneficial function of interest, said non-intergeneric geneticvariations selected from the group consisting of: full gene deletions,partial gene deletions, promoter insertions, single base pair changes,and combinations thereof (e) sequencing the genome of the candidatemicrobial species and confirming integration of the non-intergenericgenetic variation at the target genomic locus and absence of anytransgenic genetic sequence; and (0 repeating steps d)-e) one or moretimes, until a candidate microbial species has acquired an improvedability to perform the plant-beneficial function of interest.

In one aspect, provided herein is a guided microbial remodeling methodfor the rational improvement of plant-associated microbes to performplant-beneficial functions, comprising: (a) providing a plurality ofmicrobial species; (b) assaying the plurality of microbial species for:colonization metrics, transcriptionally active genes under metabolicallyrelevant environmental conditions, and ability to perform aplant-beneficial function of interest; (c) selecting a candidatemicrobial species from the plurality of assayed microbial species; (d)introducing two or more targeted non-intergeneric genetic variationsinto the candidate microbial species at two or more target genomic loci,in one or more genomic pathways, or sets of genes, that are associatedwith a plant-beneficial function of interest, said non-intergenericgenetic variations selected from the group consisting of: full genedeletions, partial gene deletions, promoter insertions, single base pairchanges, and combinations thereof; and (e) sequencing the genome of thecandidate microbial species and confirming introduction of thenon-intergeneric genetic variations at the target genomic loci andabsence of any transgenic genetic sequence.

In one aspect, provided is a computationally guided microbial remodelingmethod for the rational improvement of plant-associated microbes toperform plant-beneficial functions, comprising: (a) accessing aplurality of microbial whole genome sequences; (b) identifying aplurality of regulatory gene sequences that actively regulate thetranscription of a gene under a metabolically relevant environmentalcondition; (c) identifying a plurality of genes associated with aplant-beneficial function; (d) selecting a regulatory gene sequence anda gene associated with a plant-beneficial function from saidpluralities; wherein steps a)-d) occur in silico; and (e) manufacturing,in vivo, a remodeled microbial cell that has the selected regulatorygene sequence operably linked to the selected gene associated with aplant-beneficial function, thereby improving the expression of the geneassociated with a plant-beneficial function.

In one aspect, provided is a computationally guided microbial remodelingsystem for the rational improvement of plant-associated microbes toperform plant-beneficial functions, comprising: (a) one or moreprocessors; and (b) one or more memories operatively coupled to the oneor more processors and having instructions stored thereon, that whenexecuted by the one or more processors, cause the system to: (i) accessa plurality of microbial whole genome sequences; (ii) identify aplurality of regulatory gene sequences that actively regulate thetranscription of a gene under a metabolically relevant environmentalcondition; (iii) identify a plurality of genes associated with aplant-beneficial function; and (iv) select a regulatory gene sequenceand a gene associated with a plant-beneficial function from saidpluralities.

In one aspect, provided is a computationally guided microbial remodelingmethod for the rational improvement of plant-associated microbes toperform plant-beneficial functions, comprising: (a) activating acomputer system having: one or more processors and one or more memoriesoperatively coupled to the one or more processors and havinginstructions stored thereon, thereby causing the one or more processorsto execute the instructions, and cause the system to: (i) access aplurality of microbial whole genome sequences; (ii) identify a pluralityof regulatory gene sequences that actively regulate the transcription ofa gene under a metabolically relevant environmental condition; (iii)identify a plurality of genes associated with a plant-beneficialfunction; (iv) select a regulatory gene sequence and a gene associatedwith a plant-beneficial function from said pluralities; and (b)manufacturing, in vivo, a remodeled microbial cell that has the selectedregulatory gene sequence operably linked to the selected gene associatedwith a plant-beneficial function, thereby improving the expression ofthe gene associated with a plant-beneficial function.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts an overview of the guided microbial remodeling process,in accordance with embodiments.

FIG. 1B depicts an expanded view of the measurement of microbiomecomposition as shown in FIG. 1A.

FIG. 1C depicts a problematic “traditional bioprospecting” approach,which has several drawbacks compared to the taught guided microbialremodeling (GMR) platform.

FIG. 1D depicts a problematic “field-first approach to bioprospecting”system, which has several drawbacks compared to the taught guidedmicrobial remodeling (GMR) platform.

FIG. 1E depicts the time period in the corn growth cycle, at whichnitrogen is needed most by the plant.

FIG. 1F depicts an overview of a field development process for aremodeled microbe.

FIG. 1G depicts an overview of a guided microbial remodeling platformembodiment.

FIG. 1H depicts an overview of a computationally-guided microbialremodeling platform.

FIG. 1I depicts the use of field data combined with modeling in aspectsof the guided microbial remodeling platform.

FIG. 1J depicts 5 properties that can be possessed by remodeled microbesof the present disclosure.

FIG. 1K depicts a schematic of a remodeling approach for a microbe,PBC6.1.

FIG. 1L depicts decoupled nifA expression from endogenous nitrogenregulation in remodeled microbes.

FIG. 1M depicts improved assimilation and excretion of fixed nitrogen byremodeled microbes.

FIG. 1N depicts corn yield improvement attributable to remodeledmicrobes.

FIG. 1O illustrates the inefficiency of current nitrogen deliverysystems, which result in underfertilized fields, over fertilized fields,and environmentally deleterious nitrogen runoff

FIG. 2 illustrates PBC6.1 colonization to nearly 21% abundance of theroot-associated microbiota in corn roots. Abundance data is based on 16Samplicon sequencing of the rhizosphere and endosphere of corn plantsinoculated with PBC6.1 and grown in greenhouse conditions.

FIGS. 3A-3E illustrate derivative microbes that fix and excrete nitrogenin vitro under conditions similar to high nitrate agricultural soils.FIG. 3A illustrates the regulatory network controlling nitrogen fixationand assimilation in PBC6.1 is shown, including the key nodes NifL, NifA,GS, GlnE depicted as the two-domain ATase-AR enzyme, and AmtB. FIG. 3Billustrates the genome of Kosakonia sacchari isolate PBC6.1. The threetracks circumscribing the genome convey transcription data from PBC6.1,PBC6.38, and the differential expression between the strainsrespectively. FIG. 3C illustrates the nitrogen fixation gene cluster andtranscription data is expanded for finer detail. FIG. 3D illustratesnitrogenase activity under varying concentrations of exogenous nitrogenis measured with the acetylene reduction assay. The wild type strainexhibits repression of nitrogenase activity as glutamine concentrationsincrease, while derivative strains show varying degrees of robustness.In the line graph, triangles represent strain PBC6.22; circles representstrain PBC6.1; squares represent strain PBC6.15; and diamonds representstrain PBC6.14. Error bars represent standard error of the mean of atleast three biological replicates. FIG. 3E illustrates temporalexcretion of ammonia by derivative strains is observed at mMconcentrations. Wild type strains are not observed to excrete fixednitrogen, and negligible ammonia accumulates in the media. Error barsrepresent standard error of the mean.

FIG. 4 illustrates transcriptional rates of nifA in derivative strainsof PBC6.1 correlated with acetylene reduction rates. An ARA assay wasperformed as described in the Methods, after which cultures were sampledand subjected to qPCR analysis to determine nifA transcript levels.Error bars show standard error of the mean of at least three biologicalreplicates in each measure.

FIGS. 5A-5C illustrate greenhouse experiments that demonstrate microbialnitrogen fixation in corn. FIG. 5A illustrates microbe colonization sixweeks after inoculation of corn plants by PBC6.1 derivative strains.Error bars show standard error of the mean of at least eight biologicalreplicates. FIG. 5B illustrates in planta transcription of nifH measuredby extraction of total RNA from roots and subsequent Nanostringanalysis. Only derivative strains show nifH transcription in the rootenvironment. Error bars show standard error of the mean of at least 3biological replicates. FIG. 5C illustrates microbial nitrogen fixationmeasured by the dilution of isotopic tracer in plant tissues. Derivativemicrobes exhibit substantial transfer of fixed nitrogen to the plant.Error bars show standard error of the mean of at least ten biologicalreplicates.

FIG. 6 depicts the lineage of modified strains that were derived fromstrain C1006.

FIG. 7 depicts the lineage of modified strains that were derived fromstrain C1019.

FIG. 8 depicts a heatmap of the pounds of nitrogen delivered peracre-season by microbes of the present disclosure recorded as a functionof microbes per g-fresh weight by mmol of nitrogen/microbe-hr. Below thethin line that transects the larger image are the microbes that deliverless than one pound of nitrogen per acre-season, and above the line arethe microbes that deliver greater than one pound of nitrogen peracre-season. The table below the heatmap gives the precise value of mmolN produced per microbe per hour (mmol N/Microbe hr) along with theprecise CFU per gram of fresh weight (CFU/g fw) for each microbe shownin the heatmap. The microbes utilized in the heatmap were assayed for Nproduction in corn. For the WT strains C1006 and C1019, corn rootcolonization data was taken from a single field site. For the remainingstrains, colonization was assumed to be the same as the WT field level.N-fixation activity was determined using an in vitro ARA assay at 5 mMglutamine.

FIG. 9 depicts the plant yield of plants having been exposed to strainC1006. The area of the circles corresponds to the relative yield, whilethe shading corresponds to the particular MRTN treatment. The x-axis isthe p value and the y-axis is the win rate.

FIG. 10 depicts the plant yield of plants having been exposed to strainCM029. The area of the circles corresponds to the relative yield, whilethe shading corresponds to the particular MRTN treatment. The x-axis isthe p value and the y-axis is the win rate.

FIG. 11 depicts the plant yield of plants having been exposed to strainCM038. The area of the circles corresponds to the relative yield, whilethe shading corresponds to the particular MRTN treatment. The x-axis isthe p value and the y-axis is the win rate.

FIG. 12 depicts the plant yield of plants having been exposed to strainCI019. The area of the circles corresponds to the relative yield, whilethe shading corresponds to the particular MRTN treatment. The x-axis isthe p value and the y-axis is the win rate.

FIG. 13 depicts the plant yield of plants having been exposed to strainCM081. The area of the circles corresponds to the relative yield, whilethe shading corresponds to the particular MRTN treatment. The x-axis isthe p value and the y-axis is the win rate.

FIG. 14 depicts the plant yield of plants having been exposed to strainsCM029 and CM081. The area of the circles corresponds to the relativeyield, while the shading corresponds to the particular MRTN treatment.The x-axis is the p value and the y-axis is the win rate.

FIG. 15 depicts the plant yield of plants as the aggregated bushelgain/loss. The area of the circles corresponds to the relative yield,while the shading corresponds to the particular MRTN treatment. Thex-axis is the p value and the y-axis is the win rate.

FIG. 16 illustrates results from a summer 2017 field testing experiment.The yield results obtained demonstrate that the microbes of thedisclosure can serve as a potential fertilizer replacement. Forinstance, the utilization of a microbe of the disclosure (i.e. 6-403)resulted in a higher yield than the wild type strain (WT) and a higheryield than the untreated control (UTC). The “−25 lbs N” treatmentutilizes 25 lbs less N per acre than standard agricultural practices ofthe region. The “100% N” UTC treatment is meant to depict standardagricultural practices of the region, in which 100% of the standardutilization of N is deployed by the farmer. The microbe “6-403” wasdeposited as NCMA 201708004 and can be found in Table 1. This is amutant Kosakonia sacchari (also called CM037) and is a progeny mutantstrain from CI006 WT.

FIG. 17 illustrates results from a summer 2017 field testing experiment.The yield results obtained demonstrate that the microbes of thedisclosure perform consistently across locations. Furthermore, the yieldresults demonstrate that the microbes of the disclosure perform well inboth a nitrogen stressed environment, as well as an environment that hassufficient supplies of nitrogen. The microbe “6-881” (also known asCM094, PBC6.94), and which is a progeny mutant Kosakonia sacchari strainfrom CI006 WT, was deposited as NCMA 201708002 and can be found inTable 1. The microbe “137-1034,” which is a progeny mutant Klebsiellavariicola strain from CI137 WT, was deposited as NCMA 201712001 and canbe found in Table 1. The microbe “137-1036,” which is a progeny mutantKlebsiella variicola strain from CI137 WT, was deposited as NCMA201712002 and can be found in Table 1. The microbe “6-404” (also knownas CM38, PBC6.38), and which is a progeny mutant Kosakonia saccharistrain from CI006 WT, was deposited as NCMA 201708003 and can be foundin Table 1. The “Nutrient Stress” condition corresponds to the 0%nitrogen regime. The “Sufficient Fertilizer” condition corresponds tothe 100% nitrogen regime.

FIG. 18 depicts the lineage of modified strains that were derived fromstrain CI006 (also termed “6”, Kosakonia sacchari WT).

FIG. 19 depicts the lineage of modified strains that were derived fromstrain CI019 (also termed “19”, Rahnella aquatilis WT).

FIG. 20 depicts the lineage of modified strains that were derived fromstrain CI137 (also termed (“137”, Klebsiella variicola WT).

FIG. 21 depicts the lineage of modified strains that were derived fromstrain 1021 (Kosakonia pseudosacchari WT).

FIG. 22 depicts the lineage of modified strains that were derived fromstrain 910 (Kluyvera intermedia WT).

FIG. 23 depicts the lineage of modified strains that were derived fromstrain 63 (Rahnella aquatilis WT).

FIG. 24 depicts a heatmap of the pounds of nitrogen delivered peracre-season by microbes of the present disclosure recorded as a functionof microbes per g-fresh weight by mmol of nitrogen/microbe-hr. Below thethin line that transects the larger image are the microbes that deliverless than one pound of nitrogen per acre-season, and above the line arethe microbes that deliver greater than one pound of nitrogen peracre-season. The Table 28 in Example 5 gives the precise value of mmol Nproduced per microbe per hour (mmol N/Microbe hr) along with the preciseCFU per gram of fresh weight (CFU/g fw) for each microbe shown in theheatmap. The data in FIG. 24 is derived from microbial strains assayedfor N production in corn in field conditions. Each point represents lbN/acre produced by a microbe using corn root colonization data from asingle field site. N-fixation activity was determined using in vitro ARAassay at 5 mM N in the form of glutamine or ammonium phosphate.

FIG. 25 depicts a heatmap of the pounds of nitrogen delivered peracre-season by microbes of the present disclosure recorded as a functionof microbes per g-fresh weight by mmol of nitrogen/microbe-hr. Below thethin line that transects the larger image are the microbes that deliverless than one pound of nitrogen per acre-season, and above the line arethe microbes that deliver greater than one pound of nitrogen peracre-season. The Table 29 in Example 5 gives the precise value of mmol Nproduced per microbe per hour (mmol N/Microbe hr) along with the preciseCFU per gram of fresh weight (CFU/g fw) for each microbe shown in theheatmap. The data in FIG. 25 is derived from microbial strains assayedfor N production in corn in laboratory and greenhouse conditions. Eachpoint represents lb N/acre produced by a single strain. White pointsrepresent strains in which corn root colonization data was gathered ingreenhouse conditions. Black points represent mutant strains for whichcorn root colonization levels are derived from average field corn rootcolonization levels of the wild-type parent strain. Hatched pointsrepresent the wild type parent strains at their average field corn rootcolonization levels. In all cases, N-fixation activity was determined byin vitro ARA assay at 5 mM N in the form of glutamine or ammoniumphosphate.

FIG. 26 illustrates excretion of ammonia excretion by WT CI137 andmutant strains described in Example 7. The WT strain was observed not toexcrete ammonia, and negligible ammonia accumulates in the media.

FIG. 27 illustrates microbial colonization in corn three weeks afterplanting. The corn seed was inoculated at planting by CI137 WT andderivative strains described in Example 7.

FIG. 28 illustrates the same data from FIG. 27 but in a slightlydifferent way. FIG. 28 illustrates average microbial colonization incorn three weeks after planting. The corn seed was inoculated atplanting by CI137 WT and derivative strains described in Example 7.

DETAILED DESCRIPTION OF THE DISCLOSURE

While various embodiments of the disclosure have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions may occur to those skilled in theart without departing from the disclosure. It should be understood thatvarious alternatives to the embodiments of the disclosure describedherein may be employed.

Increased fertilizer utilization brings with it environmental concernsand is also likely not possible for many economically stressed regionsof the globe. Furthermore, many industry players in the microbial arenaare focused on creating intergeneric microbes. However, there is a heavyregulatory burden placed on engineered microbes that arecharacterized/classified as intergeneric. These intergeneric microbesface not only a higher regulatory burden, which makes widespreadadoption and implementation difficult, but they also face a great dealof public perception scrutiny.

Currently, there are no engineered microbes on the market that arenon-intergeneric and that are capable of increasing nitrogen fixation innon-leguminous crops. This dearth of such a microbe is a missing elementin helping to usher in a truly environmentally friendly and moresustainable 21^(st) century agricultural system.

The present disclosure solves the aforementioned problems and provides anon-intergeneric microbe that has been engineered to readily fixnitrogen in crops. These microbes are not characterized/classified asintergeneric microbes and thus will not face the steep regulatoryburdens of such. Further, the taught non-intergeneric microbes willserve to help 21^(st) century farmers become less dependent uponutilizing ever increasing amounts of exogenous nitrogen fertilizer.

Definitions

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the disclosure (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the disclosure and does not pose a limitation on the scope ofthe disclosure unless otherwise claimed. No language in thespecification should be construed as indicating any non-claimed elementas essential to the practice of the disclosure.

The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”,“nucleic acid” and “oligonucleotide” are used interchangeably. Theyrefer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three-dimensional structure, and mayperform any function, known or unknown. The following are non-limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA(rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA),micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides,branched polynucleotides, plasmids, vectors, isolated DNA of anysequence, isolated RNA of any sequence, nucleic acid probes, andprimers. A polynucleotide may comprise one or more modified nucleotides,such as methylated nucleotides and nucleotide analogs. If present,modifications to the nucleotide structure may be imparted before orafter assembly of the polymer. The sequence of nucleotides may beinterrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogstein binding, or inany other sequence specific manner according to base complementarity.The complex may comprise two strands forming a duplex structure, threeor more strands forming a multi stranded complex, a singleself-hybridizing strand, or any combination of these. A hybridizationreaction may constitute a step in a more extensive process, such as theinitiation of PCR, or the enzymatic cleavage of a polynucleotide by anendonuclease. A second sequence that is complementary to a firstsequence is referred to as the “complement” of the first sequence. Theterm “hybridizable” as applied to a polynucleotide refers to the abilityof the polynucleotide to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues in a hybridizationreaction.

“Complementarity” refers to the ability of a nucleic acid to formhydrogen bond(s) with another nucleic acid sequence by eithertraditional Watson-Crick or other non-traditional types. A percentcomplementarity indicates the percentage of residues in a nucleic acidmolecule which can form hydrogen bonds (e.g., Watson-Crick base pairing)with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively).“Perfectly complementary” means that all the contiguous residues of anucleic acid sequence will hydrogen bond with the same number ofcontiguous residues in a second nucleic acid sequence. “Substantiallycomplementary” as used herein refers to a degree of complementarity thatis at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refersto two nucleic acids that hybridize under stringent conditions. Sequenceidentity, such as for the purpose of assessing percent complementarity,may be measured by any suitable alignment algorithm, including but notlimited to the Needleman-Wunsch algorithm (see e.g. the EMBOSS Needlealigner available atwww.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html, optionally withdefault settings), the BLAST algorithm (see e.g. the BLAST alignmenttool available at blast.ncbi.nlm.nih.gov/Blast.cgi, optionally withdefault settings), or the Smith-Waterman algorithm (see e.g. the EMBOSSWater aligner available atwww.ebi.ac.uk/Tools/psa/emboss_water/nucleotide.html, optionally withdefault settings). Optimal alignment may be assessed using any suitableparameters of a chosen algorithm, including default parameters.

In general, “stringent conditions” for hybridization refer to conditionsunder which a nucleic acid having complementarity to a target sequencepredominantly hybridizes with a target sequence, and substantially doesnot hybridize to non-target sequences. Stringent conditions aregenerally sequence-dependent and vary depending on a number of factors.In general, the longer the sequence, the higher the temperature at whichthe sequence specifically hybridizes to its target sequence.Non-limiting examples of stringent conditions are described in detail inTijssen (1993), Laboratory Techniques In Biochemistry And MolecularBiology-Hybridization With Nucleic Acid Probes Part I, Second Chapter“Overview of principles of hybridization and the strategy of nucleicacid probe assay”, Elsevier, N.Y.

As used herein, “expression” refers to the process by which apolynucleotide is transcribed from a DNA template (such as into and mRNAor other RNA transcript) and/or the process by which a transcribed mRNAis subsequently translated into peptides, polypeptides, or proteins.Transcripts and encoded polypeptides may be collectively referred to as“gene product.” If the polynucleotide is derived from genomic DNA,expression may include splicing of the mRNA in a eukaryotic cell.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. As used herein the term “aminoacid” includes natural and/or unnatural or synthetic amino acids,including glycine and both the D or L optical isomers, and amino acidanalogs and peptidomimetics.

As used herein, the term “about” is used synonymously with the term“approximately.” Illustratively, the use of the term “about” with regardto an amount indicates that values slightly outside the cited values,e.g., plus or minus 0.1% to 10%.

The term “biologically pure culture” or “substantially pure culture”refers to a culture of a bacterial species described herein containingno other bacterial species in quantities sufficient to interfere withthe replication of the culture or be detected by normal bacteriologicaltechniques.

“Plant productivity” refers generally to any aspect of growth ordevelopment of a plant that is a reason for which the plant is grown.For food crops, such as grains or vegetables, “plant productivity” canrefer to the yield of grain or fruit harvested from a particular crop.As used herein, improved plant productivity refers broadly toimprovements in yield of grain, fruit, flowers, or other plant partsharvested for various purposes, improvements in growth of plant parts,including stems, leaves and roots, promotion of plant growth,maintenance of high chlorophyll content in leaves, increasing fruit orseed numbers, increasing fruit or seed unit weight, reducing NO₂emission due to reduced nitrogen fertilizer usage and similarimprovements of the growth and development of plants.

Microbes in and around food crops can influence the traits of thosecrops. Plant traits that may be influenced by microbes include: yield(e.g., grain production, biomass generation, fruit development, flowerset); nutrition (e.g., nitrogen, phosphorus, potassium, iron,micronutrient acquisition); abiotic stress management (e.g., droughttolerance, salt tolerance, heat tolerance); and biotic stress management(e.g., pest, weeds, insects, fungi, and bacteria). Strategies foraltering crop traits include: increasing key metabolite concentrations;changing temporal dynamics of microbe influence on key metabolites;linking microbial metabolite production/degradation to new environmentalcues; reducing negative metabolites; and improving the balance ofmetabolites or underlying proteins.

As used herein, a “control sequence” refers to an operator, promoter,silencer, or terminator.

As used herein, “in planta” may refer to in the plant, on the plant, orintimately associated with the plant, depending upon context of usage(e.g. endophytic, epiphytic, or rhizospheric associations). The plantmay comprise plant parts, tissue, leaves, roots, root hairs, rhizomes,stems, seed, ovules, pollen, flowers, fruit, etc.

In some embodiments, native or endogenous control sequences of genes ofthe present disclosure are replaced with one or more intragenericcontrol sequences.

As used herein, “introduced” refers to the introduction by means ofmodern biotechnology, and not a naturally occurring introduction.

In some embodiments, the bacteria of the present disclosure have beenmodified such that they are not naturally occurring bacteria.

In some embodiments, the bacteria of the present disclosure are presentin the plant in an amount of at least 10³ cfu, 10⁴ cfu, 10⁵ cfu, 10⁶cfu, 10⁷ cfu, 10⁸ cfu, 10⁹ cfu, 10¹⁰ cfu, 10¹¹ cfu, or 10¹² cfu per gramof fresh or dry weight of the plant. In some embodiments, the bacteriaof the present disclosure are present in the plant in an amount of atleast about 10³ cfu, about 10⁴ cfu, about 10⁵ cfu, about 10⁶ cfu, about10⁷ cfu, about 10⁸ cfu, about 10⁹ cfu, about 10¹⁰ cfu, about 10¹¹ cfu,or about 10¹² cfu per gram of fresh or dry weight of the plant. In someembodiments, the bacteria of the present disclosure are present in theplant in an amount of at least 10³ to 10⁹, 10³ to 10⁷, 10³ to 10⁵, 10⁵to 10⁹, 10⁵ to 10⁷, 10⁶ to 10¹⁰, 10⁶ to 10⁷ cfu per gram of fresh or dryweight of the plant.

Fertilizers and exogenous nitrogen of the present disclosure maycomprise the following nitrogen-containing molecules: ammonium, nitrate,nitrite, ammonia, glutamine, etc. Nitrogen sources of the presentdisclosure may include anhydrous ammonia, ammonia sulfate, urea,diammonium phosphate, urea-form, monoammonium phosphate, ammoniumnitrate, nitrogen solutions, calcium nitrate, potassium nitrate, sodiumnitrate, etc.

As used herein, “exogenous nitrogen” refers to non-atmospheric nitrogenreadily available in the soil, field, or growth medium that is presentunder non-nitrogen limiting conditions, including ammonia, ammonium,nitrate, nitrite, urea, uric acid, ammonium acids, etc.

As used herein, “non-nitrogen limiting conditions” refers tonon-atmospheric nitrogen available in the soil, field, media atconcentrations greater than about 4 mM nitrogen, as disclosed by Kant etal. (2010. J. Exp. Biol. 62(4):1499-1509), which is incorporated hereinby reference.

As used herein, an “intergeneric microorganism” is a microorganism thatis formed by the deliberate combination of genetic material originallyisolated from organisms of different taxonomic genera. An “intergenericmutant” can be used interchangeably with “intergeneric microorganism”.An exemplary “intergeneric microorganism” includes a microorganismcontaining a mobile genetic element that was first identified in amicroorganism in a genus different from the recipient microorganism.Further explanation can be found, inter alia, in 40 C.F.R. § 725.3.

In aspects, microbes taught herein are “non-intergeneric,” which meansthat the microbes are not intergeneric.

As used herein, an “intrageneric microorganism” is a microorganism thatis formed by the deliberate combination of genetic material originallyisolated from organisms of the same taxonomic genera. An “intragenericmutant” can be used interchangeably with “intrageneric microorganism”.

As used herein, “introduced genetic material” means genetic materialthat is added to, and remains as a component of, the genome of therecipient.

As used herein, in the context of non-intergeneric microorganisms, theterm “remodeled” is used synonymously with the term “engineered”.Consequently, a “non-intergeneric remodeled microorganism” has asynonymous meaning to “non-intergeneric engineered microorganism,” andwill be utilized interchangeably. Further, the disclosure may refer toan “engineered strain” or “engineered derivative” or “engineerednon-intergeneric microbe,” these terms are used synonymously with“remodeled strain” or “remodeled derivative” or “remodelednon-intergeneric microbe.”

In some embodiments, the nitrogen fixation and assimilation geneticregulatory network comprises polynucleotides encoding genes andnon-coding sequences that direct, modulate, and/or regulate microbialnitrogen fixation and/or assimilation and can comprise polynucleotidesequences of the nif cluster (e.g., nifA, nijB, nifC, . . . nifZ),polynucleotides encoding nitrogen regulatory protein C, polynucleotidesencoding nitrogen regulatory protein B, polynucleotide sequences of thegln cluster (e.g. glnA and glnD), draT, and ammoniatransporters/permeases. In some cases, the Nif cluster may compriseNifB, NifH, NifD, NifK, NifE, NifN, NifX, hesa, and NifV. In some cases,the Nif cluster may comprise a subset of NifB, NifH, NifD, NifK, NifE,NifN, NifX, hesa, and NifV.

In some embodiments, fertilizer of the present disclosure comprises atleast 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% nitrogen byweight.

In some embodiments, fertilizer of the present disclosure comprises atleast about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%,about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%,about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%,about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%,about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%,about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%,about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, or about 99% nitrogen by weight.

In some embodiments, fertilizer of the present disclosure comprisesabout 5% to 50%, about 5% to 75%, about 10% to 50%, about 10% to 75%,about 15% to 50%, about 15% to 75%, about 20% to 50%, about 20% to 75%,about 25% to 50%, about 25% to 75%, about 30% to 50%, about 30% to 75%,about 35% to 50%, about 35% to 75%, about 40% to 50%, about 40% to 75%,about 45% to 50%, about 45% to 75%, or about 50% to 75% nitrogen byweight.

In some embodiments, the increase of nitrogen fixation and/or theproduction of 1% or more of the nitrogen in the plant are measuredrelative to control plants, which have not been exposed to the bacteriaof the present disclosure. All increases or decreases in bacteria aremeasured relative to control bacteria. All increases or decreases inplants are measured relative to control plants.

As used herein, a “constitutive promoter” is a promoter, which is activeunder most conditions and/or during most development stages. There areseveral advantages to using constitutive promoters in expression vectorsused in biotechnology, such as: high level of production of proteinsused to select transgenic cells or organisms; high level of expressionof reporter proteins or scorable markers, allowing easy detection andquantification; high level of production of a transcription factor thatis part of a regulatory transcription system; production of compoundsthat requires ubiquitous activity in the organism; and production ofcompounds that are required during all stages of development.Non-limiting exemplary constitutive promoters include, CaMV 35Spromoter, opine promoters, ubiquitin promoter, alcohol dehydrogenasepromoter, etc.

As used herein, a “non-constitutive promoter” is a promoter which isactive under certain conditions, in certain types of cells, and/orduring certain development stages. For example, tissue specific, tissuepreferred, cell type specific, cell type preferred, inducible promoters,and promoters under development control are non-constitutive promoters.Examples of promoters under developmental control include promoters thatpreferentially initiate transcription in certain tissues.

As used herein, “inducible” or “repressible” promoter is a promoter thatis under chemical or environmental factor's control. Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions, certain chemicals, the presenceof light, acidic or basic conditions, etc.

As used herein, a “tissue specific” promoter is a promoter thatinitiates transcription only in certain tissues. Unlike constitutiveexpression of genes, tissue-specific expression is the result of severalinteracting levels of gene regulation. As such, in the art sometimes itis preferable to use promoters from homologous or closely relatedspecies to achieve efficient and reliable expression of transgenes inparticular tissues. This is one of the main reasons for the large amountof tissue-specific promoters isolated from particular tissues found inboth scientific and patent literature.

As used herein, the term “operably linked” refers to the association ofnucleic acid sequences on a single nucleic acid fragment so that thefunction of one is regulated by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of regulatingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of thedisclosure can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

In aspects, “applying to the plant a plurality of non-intergenericbacteria,” includes any means by which the plant (including plant partssuch as a seed, root, stem, tissue, etc.) is made to come into contact(i.e. exposed) with said bacteria at any stage of the plant's lifecycle. Consequently, “applying to the plant a plurality ofnon-intergeneric bacteria,” includes any of the following means ofexposing the plant (including plant parts such as a seed, root, stem,tissue, etc.) to said bacteria: spraying onto plant, dripping ontoplant, applying as a seed coat, applying to a field that will then beplanted with seed, applying to a field already planted with seed,applying to a field with adult plants, etc.

As used herein “MRTN” is an acronym for maximum return to nitrogen andis utilized as an experimental treatment in the Examples. MRTN wasdeveloped by Iowa State University and information can be found at:cnrc.agron.iastate.edu. The MRTN is the nitrogen rate where the economicnet return to nitrogen application is maximized. The approach tocalculating the MRTN is a regional approach for developing corn nitrogenrate guidelines in individual states. The nitrogen rate trial data wasevaluated for Illinois, Iowa, Michigan, Minnesota, Ohio, and Wisconsinwhere an adequate number of research trials were available for cornplantings following soybean and corn plantings following corn. Thetrials were conducted with spring, sidedress, or splitpreplant/sidedress applied nitrogen, and sites were not irrigated exceptfor those that were indicated for irrigated sands in Wisconsin. MRTN wasdeveloped by Iowa State University due to apparent differences inmethods for determining suggested nitrogen rates required for cornproduction, misperceptions pertaining to nitrogen rate guidelines, andconcerns about application rates. By calculating the MRTN, practitionerscan determine the following: (1) the nitrogen rate where the economicnet return to nitrogen application is maximized, (2) the economicoptimum nitrogen rate, which is the point where the last increment ofnitrogen returns a yield increase large enough to pay for the additionalnitrogen, (3) the value of corn grain increase attributed to nitrogenapplication, and the maximum yield, which is the yield where applicationof more nitrogen does not result in a corn yield increase. Thus, theMRTN calculations provide practitioners with the means to maximize corncrops in different regions while maximizing financial gains fromnitrogen applications.

The term mmol is an abbreviation for millimole, which is a thousandth(10^(−s)) of a mole, abbreviated herein as mol.

As used herein the terms “microorganism” or “microbe” should be takenbroadly. These terms, used interchangeably, include but are not limitedto, the two prokaryotic domains, Bacteria and Archaea. The term may alsoencompass eukaryotic fungi and protists.

The term “microbial consortia” or “microbial consortium” refers to asubset of a microbial community of individual microbial species, orstrains of a species, which can be described as carrying out a commonfunction, or can be described as participating in, or leading to, orcorrelating with, a recognizable parameter, such as a phenotypic traitof interest.

The term “microbial community” means a group of microbes comprising twoor more species or strains. Unlike microbial consortia, a microbialcommunity does not have to be carrying out a common function, or doesnot have to be participating in, or leading to, or correlating with, arecognizable parameter, such as a phenotypic trait of interest.

As used herein, “isolate,” “isolated,” “isolated microbe,” and liketerms, are intended to mean that the one or more microorganisms has beenseparated from at least one of the materials with which it is associatedin a particular environment (for example soil, water, plant tissue,etc.). Thus, an “isolated microbe” does not exist in its naturallyoccurring environment; rather, it is through the various techniquesdescribed herein that the microbe has been removed from its naturalsetting and placed into a non-naturally occurring state of existence.Thus, the isolated strain or isolated microbe may exist as, for example,a biologically pure culture, or as spores (or other forms of thestrain). In aspects, the isolated microbe may be in association with anacceptable carrier, which may be an agriculturally acceptable carrier.

In certain aspects of the disclosure, the isolated microbes exist as“isolated and biologically pure cultures.” It will be appreciated by oneof skill in the art that an isolated and biologically pure culture of aparticular microbe denotes that said culture is substantially free ofother living organisms and contains only the individual microbe inquestion. The culture can contain varying concentrations of saidmicrobe. The present disclosure notes that isolated and biologicallypure microbes often “necessarily differ from less pure or impurematerials.” See, e.g. In re Bergstrom, 427 F.2d 1394, (CCPA1970)(discussing purified prostaglandins), see also, In re Bergy, 596F.2d 952 (CCPA 1979)(discussing purified microbes), see also,Parke-Davis & Co. v. H. K. Mulford & Co., 189 F. 95 (S.D.N.Y. 1911)(Learned Hand discussing purified adrenaline), aff'd in part, rev'd inpart, 196 F. 496 (2d Cir. 1912), each of which are incorporated hereinby reference. Furthermore, in some aspects, the disclosure provides forcertain quantitative measures of the concentration, or puritylimitations, that must be found within an isolated and biologically puremicrobial culture. The presence of these purity values, in certainembodiments, is a further attribute that distinguishes the presentlydisclosed microbes from those microbes existing in a natural state. See,e.g., Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4thCir. 1958) (discussing purity limitations for vitamin B12 produced bymicrobes), incorporated herein by reference.

As used herein, “individual isolates” should be taken to mean acomposition, or culture, comprising a predominance of a single genera,species, or strain, of microorganism, following separation from one ormore other microorganisms.

Microbes of the present disclosure may include spores and/or vegetativecells. In some embodiments, microbes of the present disclosure includemicrobes in a viable but non-culturable (VBNC) state. As used herein,“spore” or “spores” refer to structures produced by bacteria and fungithat are adapted for survival and dispersal. Spores are generallycharacterized as dormant structures; however, spores are capable ofdifferentiation through the process of germination. Germination is thedifferentiation of spores into vegetative cells that are capable ofmetabolic activity, growth, and reproduction. The germination of asingle spore results in a single fungal or bacterial vegetative cell.Fungal spores are units of asexual reproduction, and in some cases arenecessary structures in fungal life cycles. Bacterial spores arestructures for surviving conditions that may ordinarily be nonconduciveto the survival or growth of vegetative cells.

As used herein, “microbial composition” refers to a compositioncomprising one or more microbes of the present disclosure. In someembodiments, a microbial composition is administered to plants(including various plant parts) and/or in agricultural fields.

As used herein, “carrier,” “acceptable carrier,” or “agriculturallyacceptable carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the microbe can be administered, which does not detrimentallyeffect the microbe.

Regulation of Nitrogen Fixation

In some cases, nitrogen fixation pathway may act as a target for geneticengineering and optimization. One trait that may be targeted forregulation by the methods described herein is nitrogen fixation.Nitrogen fertilizer is the largest operational expense on a farm and thebiggest driver of higher yields in row crops like corn and wheat.Described herein are microbial products that can deliver renewable formsof nitrogen in non-leguminous crops. While some endophytes have thegenetics necessary for fixing nitrogen in pure culture, the fundamentaltechnical challenge is that wild-type endophytes of cereals and grassesstop fixing nitrogen in fertilized fields. The application of chemicalfertilizers and residual nitrogen levels in field soils signal themicrobe to shut down the biochemical pathway for nitrogen fixation.

Changes to the transcriptional and post-translational levels ofcomponents of the nitrogen fixation regulatory network may be beneficialto the development of a microbe capable of fixing and transferringnitrogen to corn in the presence of fertilizer. To that end, describedherein is Host-Microbe Evolution (HoME) technology to precisely evolveregulatory networks and elicit novel phenotypes. Also described hereinare unique, proprietary libraries of nitrogen-fixing endophytes isolatedfrom corn, paired with extensive omics data surrounding the interactionof microbes and host plant under different environmental conditions likenitrogen stress and excess. In some embodiments, this technology enablesprecision evolution of the genetic regulatory network of endophytes toproduce microbes that actively fix nitrogen even in the presence offertilizer in the field. Also described herein are evaluations of thetechnical potential of evolving microbes that colonize corn root tissuesand produce nitrogen for fertilized plants and evaluations of thecompatibility of endophytes with standard formulation practices anddiverse soils to determine feasibility of integrating the microbes intomodern nitrogen management strategies.

In order to utilize elemental nitrogen (N) for chemical synthesis, lifeforms combine nitrogen gas (N₂) available in the atmosphere withhydrogen in a process known as nitrogen fixation. Because of theenergy-intensive nature of biological nitrogen fixation, diazotrophs(bacteria and archaea that fix atmospheric nitrogen gas) have evolvedsophisticated and tight regulation of the nif gene cluster in responseto environmental oxygen and available nitrogen. Nif genes encode enzymesinvolved in nitrogen fixation (such as the nitrogenase complex) andproteins that regulate nitrogen fixation. Shamseldin (2013. Global J.Biotechnol. Biochem. 8(4):84-94) discloses detailed descriptions of nifgenes and their products, and is incorporated herein by reference.Described herein are methods of producing a plant with an improved traitcomprising isolating bacteria from a first plant, introducing a geneticvariation into a gene of the isolated bacteria to increase nitrogenfixation, exposing a second plant to the variant bacteria, isolatingbacteria from the second plant having an improved trait relative to thefirst plant, and repeating the steps with bacteria isolated from thesecond plant.

In Proteobacteria, regulation of nitrogen fixation centers around theσ₅₄-dependent enhancer-binding protein NifA, the positivetranscriptional regulator of the nif cluster. Intracellular levels ofactive NifA are controlled by two key factors: transcription of thenifLA operon, and inhibition of NifA activity by protein-proteininteraction with NifL. Both of these processes are responsive tointracellular glutamine levels via the PII protein signaling cascade.This cascade is mediated by GlnD, which directly senses glutamine andcatalyzes the uridylylation or deuridylylation of two PII regulatoryproteins—GlnB and GlnK—in response the absence or presence,respectively, of bound glutamine. Under conditions of nitrogen excess,unmodified GlnB signals the deactivation of the nifLA promoter. However,under conditions of nitrogen limitation, GlnB is post-translationallymodified, which inhibits its activity and leads to transcription of thenifLA operon. In this way, nifLA transcription is tightly controlled inresponse to environmental nitrogen via the PII protein signalingcascade. On the post-translational level of NifA regulation, GlnKinhibits the NifL/NifA interaction in a matter dependent on the overalllevel of free GlnK within the cell.

NifA is transcribed from the nifLA operon, whose promoter is activatedby phosphorylated NtrC, another σ₅₄-dependent regulator. Thephosphorylation state of NtrC is mediated by the histidine kinase NtrB,which interacts with deuridylylated GlnB but not uridylylated GlnB.Under conditions of nitrogen excess, a high intracellular level ofglutamine leads to deuridylylation of GlnB, which then interacts withNtrB to deactivate its phosphorylation activity and activate itsphosphatase activity, resulting in dephosphorylation of NtrC and thedeactivation of the nifLA promoter. However, under conditions ofnitrogen limitation, a low level of intracellular glutamine results inuridylylation of GlnB, which inhibits its interaction with NtrB andallows the phosphorylation of NtrC and transcription of the nifLAoperon. In this way, nifLA expression is tightly controlled in responseto environmental nitrogen via the PII protein signaling cascade. nifA,ntrB, ntrC, and glnB, are all genes that can be mutated in the methodsdescribed herein. These processes may also be responsive tointracellular or extracellular levels of ammonia, urea or nitrates.

The activity of NifA is also regulated post-translationally in responseto environmental nitrogen, most typically through NifL-mediatedinhibition of NifA activity. In general, the interaction of NifL andNifA is influenced by the PII protein signaling cascade via GlnK,although the nature of the interactions between GlnK and NifL/NifAvaries significantly between diazotrophs. In Klebsiella pneumoniae, bothforms of GlnK inhibit the NifL/NifA interaction, and the interactionbetween GlnK and NifL/NifA is determined by the overall level of freeGlnK within the cell. Under nitrogen-excess conditions, deuridylylatedGlnK interacts with the ammonium transporter AmtB, which serves to bothblock ammonium uptake by AmtB and sequester GlnK to the membrane,allowing inhibition of NifA by NifL. On the other hand, in Azotobactervinelandii, interaction with deuridylylated GlnK is required for theNifL/NifA interaction and NifA inhibition, while uridylylation of GlnKinhibits its interaction with NifL. In diazotrophs lacking the nifLgene, there is evidence that NifA activity is inhibited directly byinteraction with the deuridylylated forms of both GlnK and GlnB undernitrogen-excess conditions. In some bacteria the Nif cluster may beregulated by glnR, and further in some cases this may comprise negativeregulation. Regardless of the mechanism, post-translational inhibitionof NifA is an important regulator of the nif cluster in most knowndiazotrophs. Additionally, nifL, amtB, glnK, and glnR are genes that canbe mutated in the methods described herein.

In addition to regulating the transcription of the nif gene cluster,many diazotrophs have evolved a mechanism for the directpost-translational modification and inhibition of the nitrogenase enzymeitself, known as nitrogenase shutoff. This is mediated byADP-ribosylation of the Fe protein (NifH) under nitrogen-excessconditions, which disrupts its interaction with the MoFe protein complex(NifDK) and abolishes nitrogenase activity. DraT catalyzes theADP-ribosylation of the Fe protein and shutoff of nitrogenase, whileDraG catalyzes the removal of ADP-ribose and reactivation ofnitrogenase. As with nifLA transcription and NifA inhibition,nitrogenase shutoff is also regulated via the PII protein signalingcascade. Under nitrogen-excess conditions, deuridylylated GlnB interactswith and activates DraT, while deuridylylated GlnK interacts with bothDraG and AmtB to form a complex, sequestering DraG to the membrane.Under nitrogen-limiting conditions, the uridylylated forms of GlnB andGlnK do not interact with DraT and DraG, respectively, leading to theinactivation of DraT and the diffusion of DraG to the Fe protein, whereit removes the ADP-ribose and activates nitrogenase. The methodsdescribed herein also contemplate introducing genetic variation into thenifH, nifD, nifK, and draT genes.

Although some endophytes have the ability to fix nitrogen in vitro,often the genetics are silenced in the field by high levels of exogenouschemical fertilizers. One can decouple the sensing of exogenous nitrogenfrom expression of the nitrogenase enzyme to facilitate field-basednitrogen fixation. Improving the integral of nitrogenase activity acrosstime further serves to augment the production of nitrogen forutilization by the crop. Specific targets for genetic variation tofacilitate field-based nitrogen fixation using the methods describedherein include one or more genes selected from the group consisting ofnifA, nifL, ntrB, ntrC, glnA, glnB, glnK, draT, amtB, glnD, glnE, nifJ,nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM,nifF, nifB, and nifQ.

An additional target for genetic variation to facilitate field-basednitrogen fixation using the methods described herein is the NifAprotein. The NifA protein is typically the activator for expression ofnitrogen fixation genes. Increasing the production of NifA (eitherconstitutively or during high ammonia condition) circumvents the nativeammonia-sensing pathway. In addition, reducing the production of NifLproteins, a known inhibitor of NifA, also leads to an increased level offreely active NifA. In addition, increasing the transcription level ofthe nifAL operon (either constitutively or during high ammoniacondition) also leads to an overall higher level of NifA proteins.Elevated level of nifAL expression is achieved by altering the promoteritself or by reducing the expression of NtrB (part of ntrB and ntrCsignaling cascade that originally would result in the shutoff of nifALoperon during high nitrogen condition). High level of NifA achieved bythese or any other methods described herein increases the nitrogenfixation activity of the endophytes.

Another target for genetic variation to facilitate field-based nitrogenfixation using the methods described herein is the GlnD/GlnB/GlnK PIIsignaling cascade. The intracellular glutamine level is sensed throughthe GlnD/GlnB/GlnK PII signaling cascade. Active site mutations in GlnDthat abolish the uridylyl-removing activity of GlnD disrupt thenitrogen-sensing cascade. In addition, reduction of the GlnBconcentration short circuits the glutamine-sensing cascade. Thesemutations “trick” the cells into perceiving a nitrogen-limited state,thereby increasing the nitrogen fixation level activity. These processesmay also be responsive to intracellular or extracellular levels ofammonia, urea or nitrates.

The amtB protein is also a target for genetic variation to facilitatefield-based nitrogen fixation using the methods described herein.Ammonia uptake from the environment can be reduced by decreasing theexpression level of amtB protein. Without intracellular ammonia, theendophyte is not able to sense the high level of ammonia, preventing thedown-regulation of nitrogen fixation genes. Any ammonia that manages toget into the intracellular compartment is converted into glutamine.Intracellular glutamine level is the major currency of nitrogen sensing.Decreasing the intracellular glutamine level prevents the cells fromsensing high ammonium levels in the environment. This effect can beachieved by increasing the expression level of glutaminase, an enzymethat converts glutamine into glutamate. In addition, intracellularglutamine can also be reduced by decreasing glutamine synthase (anenzyme that converts ammonia into glutamine). In diazotrophs, fixedammonia is quickly assimilated into glutamine and glutamate to be usedfor cellular processes. Disruptions to ammonia assimilation may enablediversion of fixed nitrogen to be exported from the cell as ammonia. Thefixed ammonia is predominantly assimilated into glutamine by glutaminesynthetase (GS), encoded by glnA, and subsequently into glutamine byglutamine oxoglutarate aminotransferase (GOGAT). In some examples, glnSencodes a glutamine synthetase. GS is regulated post-translationally byGS adenylyl transferase (GlnE), a bi-functional enzyme encoded by glnEthat catalyzes both the adenylylation and de-adenylylation of GS throughactivity of its adenylyl-transferase (AT) and adenylyl-removing (AR)domains, respectively. Under nitrogen limiting conditions, glnA isexpressed, and GlnE's AR domain de-adynylylates GS, allowing it to beactive. Under conditions of nitrogen excess, glnA expression is turnedoff, and GlnE's AT domain is activated allosterically by glutamine,causing the adenylylation and deactivation of GS.

Furthermore, the draT gene may also be a target for genetic variation tofacilitate field-based nitrogen fixation using the methods describedherein. Once nitrogen fixing enzymes are produced by the cell,nitrogenase shut-off represents another level in which celldownregulates fixation activity in high nitrogen condition. Thisshut-off could be removed by decreasing the expression level of DraT.

Methods for imparting new microbial phenotypes can be performed at thetranscriptional, translational, and post-translational levels. Thetranscriptional level includes changes at the promoter (such as changingsigma factor affinity or binding sites for transcription factors,including deletion of all or a portion of the promoter) or changingtranscription terminators and attenuators. The translational levelincludes changes at the ribosome binding sites and changing mRNAdegradation signals. The post-translational level includes mutating anenzyme's active site and changing protein-protein interactions. Thesechanges can be achieved in a multitude of ways. Reduction of expressionlevel (or complete abolishment) can be achieved by swapping the nativeribosome binding site (RBS) or promoter with another with lowerstrength/efficiency. ATG start sites can be swapped to a GTG, TTG, orCTG start codon, which results in reduction in translational activity ofthe coding region. Complete abolishment of expression can be done byknocking out (deleting) the coding region of a gene. Frameshifting theopen reading frame (ORF) likely will result in a premature stop codonalong the ORF, thereby creating a non-functional truncated product.Insertion of in-frame stop codons will also similarly create anon-functional truncated product. Addition of a degradation tag at the Nor C terminal can also be done to reduce the effective concentration ofa particular gene.

Conversely, expression level of the genes described herein can beachieved by using a stronger promoter. To ensure high promoter activityduring high nitrogen level condition (or any other condition), atranscription profile of the whole genome in a high nitrogen levelcondition could be obtained and active promoters with a desiredtranscription level can be chosen from that dataset to replace the weakpromoter. Weak start codons can be swapped out with an ATG start codonfor better translation initiation efficiency. Weak ribosomal bindingsites (RBS) can also be swapped out with a different RBS with highertranslation initiation efficiency. In addition, site specificmutagenesis can also be performed to alter the activity of an enzyme.

Increasing the level of nitrogen fixation that occurs in a plant canlead to a reduction in the amount of chemical fertilizer needed for cropproduction and reduce greenhouse gas emissions (e.g., nitrous oxide).

Regulation of Colonization Potential

One trait that may be targeted for regulation by the methods describedherein is colonization potential. Accordingly, in some embodiments,pathways and genes involved in colonization may act as a target forgenetic engineering and optimization.

In some cases, exopolysaccharides may be involved in bacterialcolonization of plants. In some cases, plant colonizing microbes mayproduce a biofilm. In some cases, plant colonizing microbes secretemolecules which may assist in adhesion to the plant, or in evading aplant immune response. In some cases, plant colonizing microbes mayexcrete signaling molecules which alter the plants response to themicrobes. In some cases, plant colonizing microbes may secrete moleculeswhich alter the local microenvironment. In some cases, a plantcolonizing microbe may alter expression of genes to adapt to a plantsaid microbe is in proximity to. In some cases, a plant colonizingmicrobe may detect the presence of a plant in the local environment andmay change expression of genes in response.

In some embodiments, to improve colonization, a gene involved in apathway selected from the group consisting of: exopolysaccharideproduction, endo-polygalaturonase production, trehalose production, andglutamine conversion may be targeted for genetic engineering andoptimization.

In some embodiments, an enzyme or pathway involved in production ofexopolysaccharides may be genetically modified to improve colonization.Exemplary genes encoding an exopolysaccharide producing enzyme that maybe targeted to improve colonization include, but are not limited to,bcsii, bcsiii, and yjbE.

In some embodiments, an enzyme or pathway involved in production of afilamentous hemagglutinin may be genetically modified to improvecolonization. For example, a fhaB gene encoding a filamentoushemagglutinin may be targeted to improve colonization.

In some embodiments, an enzyme or pathway involved in production of anendo-polygalaturonase may be genetically modified to improvecolonization. For example, a pehA gene encoding an endo-polygalaturonaseprecursor may be targeted to improve colonization.

In some embodiments, an enzyme or pathway involved in production oftrehalose may be genetically modified to improve colonization. Exemplarygenes encoding a trehalose producing enzyme that may be targeted toimprove colonization include, but are not limited to, otsB and treZ.

In some embodiments, an enzyme or pathway involved in conversion ofglutamine may be genetically modified to improve colonization. Forexample, the glsA2 gene encodes a glutaminase which converts glutamineinto ammonium and glutamate. Upregulating glsA2 improves fitness byincreasing the cell's glutamate pool, thereby increasing available N tothe cells. Accordingly, in some embodiments, the glsA2 gene may betargeted to improve colonization.

In some embodiments, colonization genes selected from the groupconsisting of: bcsii, bcsiii, yjbE, fhaB, pehA, otsB, treZ, glsA2, andcombinations thereof, may be genetically modified to improvecolonization.

Colonization genes that may be targeted to improve the colonizationpotential are also described in a PCT publication, WO/2019/032926, whichis incorporated by reference herein in its entirety.

Generation of Bacterial Populations Isolation of Bacteria

Microbes useful in methods and compositions disclosed herein can beobtained by extracting microbes from surfaces or tissues of nativeplants. Microbes can be obtained by grinding seeds to isolate microbes.Microbes can be obtained by planting seeds in diverse soil samples andrecovering microbes from tissues. Additionally, microbes can be obtainedby inoculating plants with exogenous microbes and determining whichmicrobes appear in plant tissues. Non-limiting examples of plant tissuesmay include a seed, seedling, leaf, cutting, plant, bulb, or tuber.

A method of obtaining microbes may be through the isolation of bacteriafrom soils. Bacteria may be collected from various soil types. In someexample, the soil can be characterized by traits such as high or lowfertility, levels of moisture, levels of minerals, and various croppingpractices. For example, the soil may be involved in a crop rotationwhere different crops are planted in the same soil in successiveplanting seasons. The sequential growth of different crops on the samesoil may prevent disproportionate depletion of certain minerals. Thebacteria can be isolated from the plants growing in the selected soils.The seedling plants can be harvested at 2-6 weeks of growth. Forexample, at least 400 isolates can be collected in a round of harvest.Soil and plant types reveal the plant phenotype as well as theconditions, which allow for the downstream enrichment of certainphenotypes.

Microbes can be isolated from plant tissues to assess microbial traits.The parameters for processing tissue samples may be varied to isolatedifferent types of associative microbes, such as rhizopheric bacteria,epiphytes, or endophytes. The isolates can be cultured in nitrogen-freemedia to enrich for bacteria that perform nitrogen fixation.Alternatively, microbes can be obtained from global strain banks.

In planta analytics are performed to assess microbial traits. In someembodiments, the plant tissue can be processed for screening by highthroughput processing for DNA and RNA. Additionally, non-invasivemeasurements can be used to assess plant characteristics, such ascolonization. Measurements on wild microbes can be obtained on aplant-by-plant basis. Measurements on wild microbes can also be obtainedin the field using medium throughput methods. Measurements can be donesuccessively over time. Model plant system can be used including, butnot limited to, Setaria.

Microbes in a plant system can be screened via transcriptional profilingof a microbe in a plant system. Examples of screening throughtranscriptional profiling are using methods of quantitative polymerasechain reaction (qPCR), molecular barcodes for transcript detection, NextGeneration Sequencing, and microbe tagging with fluorescent markers.Impact factors can be measured to assess colonization in the greenhouseincluding, but not limited to, microbiome, abiotic factors, soilconditions, oxygen, moisture, temperature, inoculum conditions, and rootlocalization. Nitrogen fixation can be assessed in bacteria by measuring15N gas/fertilizer (dilution) with IRMS or NanoSIMS as described hereinNanoSIMS is high-resolution secondary ion mass spectrometry. TheNanoSIMS technique is a way to investigate chemical activity frombiological samples. The catalysis of reduction of oxidation reactionsthat drive the metabolism of microorganisms can be investigated at thecellular, subcellular, molecular and elemental level. NanoSIMS canprovide high spatial resolution of greater than 0.1 μm. NanoSIMS candetect the use of isotope tracers such as ¹³C, ¹⁵N, and ¹⁸O. Therefore,NanoSIMS can be used to the chemical activity nitrogen in the cell.

Automated greenhouses can be used for in planta analytics. Plant metricsin response to microbial exposure include, but are not limited to,biomass, chloroplast analysis, CCD camera, volumetric tomographymeasurements.

One way of enriching a microbe population is according to genotype. Forexample, a polymerase chain reaction (PCR) assay with a targeted primeror specific primer. Primers designed for the nifH gene can be used toidentity diazotrophs because diazotrophs express the nifH gene in theprocess of nitrogen fixation. A microbial population can also beenriched via single-cell culture-independent approaches andchemotaxis-guided isolation approaches. Alternatively, targetedisolation of microbes can be performed by culturing the microbes onselection media. Premeditated approaches to enriching microbialpopulations for desired traits can be guided by bioinformatics data andare described herein.

Enriching for Microbes with Nitrogen Fixation Capabilities UsingBioinformatics

Bioinformatics tools can be used to identify and isolate plant growthpromoting rhizobacteria (PGPRs), which are selected based on theirability to perform nitrogen fixation. Microbes with high nitrogen fixingability can promote favorable traits in plants. Bioinformatic modes ofanalysis for the identification of PGPRs include, but are not limitedto, genomics, metagenomics, targeted isolation, gene sequencing,transcriptome sequencing, and modeling.

Genomics analysis can be used to identify PGPRs and confirm the presenceof mutations with methods of Next Generation Sequencing as describedherein and microbe version control.

Metagenomics can be used to identify and isolate PGPR using a predictionalgorithm for colonization. Metadata can also be used to identify thepresence of an engineered strain in environmental and greenhousesamples.

Transcriptomic sequencing can be used to predict genotypes leading toPGPR phenotypes. Additionally, transcriptomic data is used to identifypromoters for altering gene expression. Transcriptomic data can beanalyzed in conjunction with the Whole Genome Sequence (WGS) to generatemodels of metabolism and gene regulatory networks.

Domestication of Microbes

Microbes isolated from nature can undergo a domestication processwherein the microbes are converted to a form that is geneticallytrackable and identifiable. One way to domesticate a microbe is toengineer it with antibiotic resistance. The process of engineeringantibiotic resistance can begin by determining the antibioticsensitivity in the wild type microbial strain. If the bacteria aresensitive to the antibiotic, then the antibiotic can be a good candidatefor antibiotic resistance engineering. Subsequently, an antibioticresistant gene or a counterselectable suicide vector can be incorporatedinto the genome of a microbe using recombineering methods. Acounterselectable suicide vector may consist of a deletion of the geneof interest, a selectable marker, and the counterselectable marker sacB.Counterselection can be used to exchange native microbial DNA sequenceswith antibiotic resistant genes. A medium throughput method can be usedto evaluate multiple microbes simultaneously allowing for paralleldomestication. Alternative methods of domestication include the use ofhoming nucleases to prevent the suicide vector sequences from loopingout or from obtaining intervening vector sequences.

DNA vectors can be introduced into bacteria via several methodsincluding electroporation and chemical transformations. A standardlibrary of vectors can be used for transformations. An example of amethod of gene editing is CRISPR preceded by Cas9 testing to ensureactivity of Cas9 in the microbes.

Non-Transgenic Engineering of Microbes

A microbial population with favorable traits can be obtained viadirected evolution. Direct evolution is an approach wherein the processof natural selection is mimicked to evolve proteins or nucleic acidstowards a user-defined goal. An example of direct evolution is whenrandom mutations are introduced into a microbial population, themicrobes with the most favorable traits are selected, and the growth ofthe selected microbes is continued. The most favorable traits in growthpromoting rhizobacteria (PGPRs) may be in nitrogen fixation. The methodof directed evolution may be iterative and adaptive based on theselection process after each iteration.

Plant growth promoting rhizobacteria (PGPRs) with high capability ofnitrogen fixation can be generated. The evolution of PGPRs can becarried out via the introduction of genetic variation. Genetic variationcan be introduced via polymerase chain reaction mutagenesis,oligonucleotide-directed mutagenesis, saturation mutagenesis, fragmentshuffling mutagenesis, homologous recombination, CRISPR/Cas9 systems,chemical mutagenesis, and combinations thereof. These approaches canintroduce random mutations into the microbial population. For example,mutants can be generated using synthetic DNA or RNA viaoligonucleotide-directed mutagenesis. Mutants can be generated usingtools contained on plasmids, which are later cured. Genes of interestcan be identified using libraries from other species with improvedtraits including, but not limited to, improved PGPR properties, improvedcolonization of cereals, increased oxygen sensitivity, increasednitrogen fixation, and increased ammonia excretion. Intrageneric genescan be designed based on these libraries using software such as Geneiousor Platypus design software. Mutations can be designed with the aid ofmachine learning. Mutations can be designed with the aid of a metabolicmodel. Automated design of the mutation can be done using a la Platypusand will guide RNAs for Cas-directed mutagenesis.

The intra-generic genes can be transferred into the host microbe.Additionally, reporter systems can also be transferred to the microbe.The reporter systems characterize promoters, determine thetransformation success, screen mutants, and act as negative screeningtools.

The microbes carrying the mutation can be cultured via serial passaging.A microbial colony contains a single variant of the microbe. Microbialcolonies are screened with the aid of an automated colony picker andliquid handler. Mutants with gene duplication and increased copy numberexpress a higher genotype of the desired trait.

Selection of Plant Growth Promoting Microbes Based on Nitrogen Fixation

The microbial colonies can be screened using various assays to assessnitrogen fixation. One way to measure nitrogen fixation is via a singlefermentative assay, which measures nitrogen excretion. An alternativemethod is the acetylene reduction assay (ARA) with in-line sampling overtime. ARA can be performed in high throughput plates of microtubearrays. ARA can be performed with live plants and plant tissues. Themedia formulation and media oxygen concentration can be varied in ARAassays. Another method of screening microbial variants is by usingbiosensors. The use of NanoSIMS and Raman microspectroscopy can be usedto investigate the activity of the microbes. In some cases, bacteria canalso be cultured and expanded using methods of fermentation inbioreactors. The bioreactors are designed to improve robustness ofbacteria growth and to decrease the sensitivity of bacteria to oxygen.Medium to high TP plate-based microfermentors are used to evaluateoxygen sensitivity, nutritional needs, nitrogen fixation, and nitrogenexcretion. The bacteria can also be co-cultured with competitive orbeneficial microbes to elucidate cryptic pathways. Flow cytometry can beused to screen for bacteria that produce high levels of nitrogen usingchemical, colorimetric, or fluorescent indicators. The bacteria may becultured in the presence or absence of a nitrogen source. For example,the bacteria may be cultured with glutamine, ammonia, urea or nitrates.

Guided Microbial Remodeling—An Overview

Guided microbial remodeling is a method to systematically identify andimprove the role of species within the crop microbiome. In some aspects,and according to a particular methodology of grouping/categorization,the method comprises three steps: 1) selection of candidate species bymapping plant-microbe interactions and predicting regulatory networkslinked to a particular phenotype, 2) pragmatic and predictableimprovement of microbial phenotypes through intra-species crossing ofregulatory networks and gene clusters within a microbe's genome, and 3)screening and selection of new microbial genotypes that produce desiredcrop phenotypes.

To systematically assess the improvement of strains, a model is createdthat links colonization dynamics of the microbial community to geneticactivity by key species. The model is used to predict genetic targetsfor non-intergeneric genetic remodeling (i.e. engineering the geneticarchitecture of the microbe in a non-transgenic fashion). See, FIG. 1Afor a graphical representation of an embodiment of the process.

As illustrated in FIG. 1A, rational improvement of the crop microbiomemay be used to increase soil biodiversity, tune impact of keystonespecies, and/or alter timing and expression of important metabolicpathways.

To this end, the inventors have developed a platform to identify andimprove the role of strains within the crop microbiome. In some aspects,the inventors call this process microbial breeding.

The aforementioned “Guided Microbial Remodeling” process will be furtherelaborated upon in the Examples, for instance in Example 1, entitled:“Guided Microbial Remodeling—A Platform for the Rational Improvement ofMicrobial Species for Agriculture.”

Serial Passage

Production of bacteria to improve plant traits (e.g., nitrogen fixation)can be achieved through serial passage. The production of these bacteriacan be done by selecting plants, which have a particular improved traitthat is influenced by the microbial flora, in addition to identifyingbacteria and/or compositions that are capable of imparting one or moreimproved traits to one or more plants. One method of producing abacteria to improve a plant trait includes the steps of: (a) isolatingbacteria from tissue or soil of a first plant; (b) introducing a geneticvariation into one or more of the bacteria to produce one or morevariant bacteria; (c) exposing a plurality of plants to the variantbacteria; (d) isolating bacteria from tissue or soil of one of theplurality of plants, wherein the plant from which the bacteria isisolated has an improved trait relative to other plants in the pluralityof plants; and (e) repeating steps (b) to (d) with bacteria isolatedfrom the plant with an improved trait (step (d)). Steps (b) to (d) canbe repeated any number of times (e.g., once, twice, three times, fourtimes, five times, ten times, or more) until the improved trait in aplant reaches a desired level. Further, the plurality of plants can bemore than two plants, such as 10 to 20 plants, or 20 or more, 50 ormore, 100 or more, 300 or more, 500 or more, or 1000 or more plants.

In addition to obtaining a plant with an improved trait, a bacterialpopulation comprising bacteria comprising one or more genetic variationsintroduced into one or more genes (e.g., genes regulating nitrogenfixation) is obtained. By repeating the steps described above, apopulation of bacteria can be obtained that include the most appropriatemembers of the population that correlate with a plant trait of interest.The bacteria in this population can be identified and their beneficialproperties determined, such as by genetic and/or phenotypic analysis.Genetic analysis may occur of isolated bacteria in step (a). Phenotypicand/or genotypic information may be obtained using techniques including:high through-put screening of chemical components of plant origin,sequencing techniques including high throughput sequencing of geneticmaterial, differential display techniques (including DDRT-PCR, andDD-PCR), nucleic acid microarray techniques, RNA-sequencing (WholeTranscriptome Shotgun Sequencing), and qRT-PCR (quantitative real timePCR). Information gained can be used to obtain community profilinginformation on the identity and activity of bacteria present, such asphylogenetic analysis or microarray-based screening of nucleic acidscoding for components of rRNA operons or other taxonomically informativeloci. Examples of taxonomically informative loci include 16S rRNA gene,23S rRNA gene, 5S rRNA gene, 5.8S rRNA gene, 12S rRNA gene, 18S rRNAgene, 28S rRNA gene, gyrB gene, rpoB gene, fusA gene, recA gene, coxlgene, nifD gene. Example processes of taxonomic profiling to determinetaxa present in a population are described in US20140155283. Bacterialidentification may comprise characterizing activity of one or more genesor one or more signaling pathways, such as genes associated with thenitrogen fixation pathway. Synergistic interactions (where twocomponents, by virtue of their combination, increase a desired effect bymore than an additive amount) between different bacterial species mayalso be present in the bacterial populations.

Genetic Variation—Locations and Sources of Genomic Alteration

The genetic variation may be a variation in a gene selected from thegroup consisting of: nifA, nifL, ntrB, ntrC, glnA, glnB, glnK, draT,amtB, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS,nifV, nifW, nifZ, nifM, nifF, nifB, and nifQ. The genetic variation maybe a variation in a gene encoding a protein with functionality selectedfrom the group consisting of: glutamine synthetase, glutaminase,glutamine synthetase adenylyltransferase, transcriptional activator,anti-transcriptional activator, pyruvate flavodoxin oxidoreductase,flavodoxin, or NAD+-dinitrogen-reductase aDP-D-ribosyltransferase. Thegenetic variation may be a mutation that results in one or more of:increased expression or activity of NifA or glutaminase; decreasedexpression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK,DraT, AmtB; decreased adenylyl-removing activity of GlnE; or decreaseduridylyl-removing activity of GlnD. The genetic variation may be avariation in a gene selected from the group consisting of: bcsii,bcsiii, yjbE, fhaB, pehA, otsB, treZ, glsA2, and combinations thereof.In some embodiments, a genetic variation may be a variation in any ofthe genes described throughout this disclosure.

Introducing a genetic variation may comprise insertion and/or deletionof one or more nucleotides at a target site, such as 1, 2, 3, 4, 5, 10,25, 50, 100, 250, 500, or more nucleotides. The genetic variationintroduced into one or more bacteria of the methods disclosed herein maybe a knock-out mutation (e.g. deletion of a promoter, insertion ordeletion to produce a premature stop codon, deletion of an entire gene),or it may be elimination or abolishment of activity of a protein domain(e.g. point mutation affecting an active site, or deletion of a portionof a gene encoding the relevant portion of the protein product), or itmay alter or abolish a regulatory sequence of a target gene. One or moreregulatory sequences may also be inserted, including heterologousregulatory sequences and regulatory sequences found within a genome of abacterial species or genus corresponding to the bacteria into which thegenetic variation is introduced. Moreover, regulatory sequences may beselected based on the expression level of a gene in a bacterial cultureor within a plant tissue. The genetic variation may be a pre-determinedgenetic variation that is specifically introduced to a target site. Thegenetic variation may be a random mutation within the target site. Thegenetic variation may be an insertion or deletion of one or morenucleotides. In some cases, a plurality of different genetic variations(e.g. 2, 3, 4, 5, 10, or more) are introduced into one or more of theisolated bacteria before exposing the bacteria to plants for assessingtrait improvement. The plurality of genetic variations can be any of theabove types, the same or different types, and in any combination. Insome cases, a plurality of different genetic variations are introducedserially, introducing a first genetic variation after a first isolationstep, a second genetic variation after a second isolation step, and soforth so as to accumulate a plurality of genetic variations in bacteriaimparting progressively improved traits on the associated plants.

Genetic Variation—Methods of Introducing Genomic Alteration

In general, the term “genetic variation” refers to any change introducedinto a polynucleotide sequence relative to a reference polynucleotide,such as a reference genome or portion thereof, or reference gene orportion thereof. A genetic variation may be referred to as a “mutation,”and a sequence or organism comprising a genetic variation may bereferred to as a “genetic variant” or “mutant”. Genetic variations canhave any number of effects, such as the increase or decrease of somebiological activity, including gene expression, metabolism, and cellsignaling. Genetic variations can be specifically introduced to a targetsite, or introduced randomly. A variety of molecular tools and methodsare available for introducing genetic variation. For example, geneticvariation can be introduced via polymerase chain reaction mutagenesis,oligonucleotide-directed mutagenesis, saturation mutagenesis, fragmentshuffling mutagenesis, homologous recombination, recombineering, lambdared mediated recombination, CRISPR/Cas9 systems, chemical mutagenesis,and combinations thereof. Chemical methods of introducing geneticvariation include exposure of DNA to a chemical mutagen, e.g., ethylmethanesulfonate (EMS), methyl methanesulfonate (MMS), N-nitrosourea (ENU), N-methyl-N-nitro-N′-nitrosoguanidine, 4-nitroquinoline N-oxide,diethylsulfate, benzopyrene, cyclophosphamide, bleomycin,triethylmelamine, acrylamide monomer, nitrogen mustard, vincristine,diepoxyalkanes (for example, diepoxybutane), ICR-170, formaldehyde,procarbazine hydrochloride, ethylene oxide, dimethylnitrosamine, 7,12dimethylbenz(a)anthracene, chlorambucil, hexamethylphosphoramide,bisulfan, and the like. Radiation mutation-inducing agents includeultraviolet radiation, γ-irradiation, X-rays, and fast neutronbombardment. Genetic variation can also be introduced into a nucleicacid using, e.g., trimethylpsoralen with ultraviolet light. Random ortargeted insertion of a mobile DNA element, e.g., a transposableelement, is another suitable method for generating genetic variation.Genetic variations can be introduced into a nucleic acid duringamplification in a cell-free in vitro system, e.g., using a polymerasechain reaction (PCR) technique such as error-prone PCR. Geneticvariations can be introduced into a nucleic acid in vitro using DNAshuffling techniques (e.g., exon shuffling, domain swapping, and thelike). Genetic variations can also be introduced into a nucleic acid asa result of a deficiency in a DNA repair enzyme in a cell, e.g., thepresence in a cell of a mutant gene encoding a mutant DNA repair enzymeis expected to generate a high frequency of mutations (i.e., about 1mutation/100 genes-1 mutation/10,000 genes) in the genome of the cell.Examples of genes encoding DNA repair enzymes include but are notlimited to Mut H, Mut S, Mut L, and Mut U, and the homologs thereof inother species (e.g., MSH 1 6, PMS 1 2, MLH 1, GTBP, ERCC-1, and thelike). Example descriptions of various methods for introducing geneticvariations are provided in e.g., Stemple (2004) Nature 5:1-7; Chiang etal. (1993) PCR Methods Appl 2(3): 210-217; Stemmer (1994) Proc. Natl.Acad. Sci. USA 91:10747-10751; and U.S. Pat. Nos. 6,033,861, and6,773,900.

Genetic variations introduced into microbes may be classified astransgenic, cisgenic, intragenomic, intrageneric, intergeneric,synthetic, evolved, rearranged, or SNPs.

Genetic variation may be introduced into numerous metabolic pathwayswithin microbes to elicit improvements in the traits described above.Representative pathways include sulfur uptake pathways, glycogenbiosynthesis, the glutamine regulation pathway, the molybdenum uptakepathway, the nitrogen fixation pathway, ammonia assimilation, ammoniaexcretion or secretion, Nitrogen uptake, glutamine biosynthesis,annamox, phosphate solubilization, organic acid transport, organic acidproduction, agglutinins production, reactive oxygen radical scavenginggenes, Indole Acetic Acid biosynthesis, trehalose biosynthesis, plantcell wall degrading enzymes or pathways, root attachment genes,exopolysaccharide secretion, glutamate synthase pathway, iron uptakepathways, siderophore pathway, chitinase pathway, ACC deaminase,glutathione biosynthesis, phosphorous signaling genes, quorum quenchingpathway, cytochrome pathways, hemoglobin pathway, bacterialhemoglobin-like pathway, small RNA rsmZ, rhizobitoxine biosynthesis,lapA adhesion protein, AHL quorum sensing pathway, phenazinebiosynthesis, cyclic lipopeptide biosynthesis, and antibioticproduction.

CRISPR/Cas9 (Clustered regularly interspaced short palindromicrepeats)/CRISPR-associated (Cas) systems can be used to introducedesired mutations. CRISPR/Cas9 provide bacteria and archaea withadaptive immunity against viruses and plasmids by using CRISPR RNAs(crRNAs) to guide the silencing of invading nucleic acids. The Cas9protein (or functional equivalent and/or variant thereof, i.e.,Cas9-like protein) naturally contains DNA endonuclease activity thatdepends on the association of the protein with two naturally occurringor synthetic RNA molecules called crRNA and tracrRNA (also called guideRNAs). In some cases, the two molecules are covalently link to form asingle molecule (also called a single guide RNA (“sgRNA”). Thus, theCas9 or Cas9-like protein associates with a DNA-targeting RNA (whichterm encompasses both the two-molecule guide RNA configuration and thesingle-molecule guide RNA configuration), which activates the Cas9 orCas9-like protein and guides the protein to a target nucleic acidsequence. If the Cas9 or Cas9-like protein retains its natural enzymaticfunction, it will cleave target DNA to create a double-stranded break,which can lead to genome alteration (i.e., editing, deletion, insertion(when a donor polynucleotide is present), replacement, etc.), therebyaltering gene expression. Some variants of Cas9 (which variants areencompassed by the term Cas9-like) have been altered such that they havea decreased DNA cleaving activity (in some cases, they cleave a singlestrand instead of both strands of the target DNA, while in other cases,they have severely reduced to no DNA cleavage activity). Furtherexemplary descriptions of CRISPR systems for introducing geneticvariation can be found in, e.g. U.S. Pat. No. 8,795,965.

As a cyclic amplification technique, polymerase chain reaction (PCR)mutagenesis uses mutagenic primers to introduce desired mutations. PCRis performed by cycles of denaturation, annealing, and extension. Afteramplification by PCR, selection of mutated DNA and removal of parentalplasmid DNA can be accomplished by: 1) replacement of dCTP byhydroxymethylated-dCTP during PCR, followed by digestion withrestriction enzymes to remove non-hydroxymethylated parent DNA only; 2)simultaneous mutagenesis of both an antibiotic resistance gene and thestudied gene changing the plasmid to a different antibiotic resistance,the new antibiotic resistance facilitating the selection of the desiredmutation thereafter; 3) after introducing a desired mutation, digestionof the parent methylated template DNA by restriction enzyme Dpn1 whichcleaves only methylated DNA, by which the mutagenized unmethylatedchains are recovered; or 4) circularization of the mutated PCR productsin an additional ligation reaction to increase the transformationefficiency of mutated DNA. Further description of exemplary methods canbe found in e.g. U.S. Pat. Nos. 7,132,265, 6,713,285, 6,673,610,6,391,548, 5,789,166, 5,780,270, 5,354,670, 5,071,743, andUS20100267147.

Oligonucleotide-directed mutagenesis, also called site-directedmutagenesis, typically utilizes a synthetic DNA primer. This syntheticprimer contains the desired mutation and is complementary to thetemplate DNA around the mutation site so that it can hybridize with theDNA in the gene of interest. The mutation may be a single base change (apoint mutation), multiple base changes, deletion, or insertion, or acombination of these. The single-strand primer is then extended using aDNA polymerase, which copies the rest of the gene. The gene thus copiedcontains the mutated site, and may then be introduced into a host cellas a vector and cloned. Finally, mutants can be selected by DNAsequencing to check that they contain the desired mutation.

Genetic variations can be introduced using error-prone PCR. In thistechnique, the gene of interest is amplified using a DNA polymeraseunder conditions that are deficient in the fidelity of replication ofsequence. The result is that the amplification products contain at leastone error in the sequence. When a gene is amplified and the resultingproduct(s) of the reaction contain one or more alterations in sequencewhen compared to the template molecule, the resulting products aremutagenized as compared to the template. Another means of introducingrandom mutations is exposing cells to a chemical mutagen, such asnitrosoguanidine or ethyl methanesulfonate (Nestmann, Mutat Res 1975June; 28(3):323-30), and the vector containing the gene is then isolatedfrom the host.

Saturation mutagenesis is another form of random mutagenesis, in whichone tries to generate all or nearly all possible mutations at a specificsite, or narrow region of a gene. In a general sense, saturationmutagenesis is comprised of mutagenizing a complete set of mutageniccassettes (wherein each cassette is, for example, 1-500 bases in length)in defined polynucleotide sequence to be mutagenized (wherein thesequence to be mutagenized is, for example, from 15 to 100,000 bases inlength). Therefore, a group of mutations (e.g. ranging from 1 to 100mutations) is introduced into each cassette to be mutagenized. Agrouping of mutations to be introduced into one cassette can bedifferent or the same from a second grouping of mutations to beintroduced into a second cassette during the application of one round ofsaturation mutagenesis. Such groupings are exemplified by deletions,additions, groupings of particular codons, and groupings of particularnucleotide cassettes.

Fragment shuffling mutagenesis, also called DNA shuffling, is a way torapidly propagate beneficial mutations. In an example of a shufflingprocess, DNAse is used to fragment a set of parent genes into pieces ofe.g. about 50-100 bp in length. This is then followed by a polymerasechain reaction (PCR) without primers—DNA fragments with sufficientoverlapping homologous sequence will anneal to each other and are thenbe extended by DNA polymerase. Several rounds of this PCR extension areallowed to occur, after some of the DNA molecules reach the size of theparental genes. These genes can then be amplified with another PCR, thistime with the addition of primers that are designed to complement theends of the strands. The primers may have additional sequences added totheir 5′ ends, such as sequences for restriction enzyme recognitionsites needed for ligation into a cloning vector. Further examples ofshuffling techniques are provided in US20050266541.

Homologous recombination mutagenesis involves recombination between anexogenous DNA fragment and the targeted polynucleotide sequence. After adouble-stranded break occurs, sections of DNA around the 5′ ends of thebreak are cut away in a process called resection. In the strand invasionstep that follows, an overhanging 3′ end of the broken DNA molecule then“invades” a similar or identical DNA molecule that is not broken. Themethod can be used to delete a gene, remove exons, add a gene, andintroduce point mutations. Homologous recombination mutagenesis can bepermanent or conditional. Typically, a recombination template is alsoprovided. A recombination template may be a component of another vector,contained in a separate vector, or provided as a separatepolynucleotide. In some embodiments, a recombination template isdesigned to serve as a template in homologous recombination, such aswithin or near a target sequence nicked or cleaved by a site-specificnuclease. A template polynucleotide may be of any suitable length, suchas about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500,1000, or more nucleotides in length. In some embodiments, the templatepolynucleotide is complementary to a portion of a polynucleotidecomprising the target sequence. When optimally aligned, a templatepolynucleotide might overlap with one or more nucleotides of a targetsequences (e.g. about or more than about 1, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides). In someembodiments, when a template sequence and a polynucleotide comprising atarget sequence are optimally aligned, the nearest nucleotide of thetemplate polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75,100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from thetarget sequence. Non-limiting examples of site-directed nucleases usefulin methods of homologous recombination include zinc finger nucleases,CRISPR nucleases, TALE nucleases, and meganuclease. For a furtherdescription of the use of such nucleases, see e.g. U.S. Pat. No.8,795,965 and US20140301990.

Mutagens that create primarily point mutations and short deletions,insertions, transversions, and/or transitions, including chemicalmutagens or radiation, may be used to create genetic variations.Mutagens include, but are not limited to, ethyl methanesulfonate,methylmethane sulfonate, N-ethyl-N-nitrosourea, triethylmelamine,N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide,diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard,vincristine, dimethylnitrosamine, N-methyl-N′-nitro-Nitrosoguanidine,nitrosoguanidine, 2-aminopurine, 7,12 dimethyl-benz(a)anthracene,ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes(diepoxyoctane, diepoxybutane, and the like),2-methoxy-6-chloro-9[3-(ethyl-2-chloro-ethyl)aminopropylamino]acridinedihydrochloride and formaldehyde.

Introducing genetic variation may be an incomplete process, such thatsome bacteria in a treated population of bacteria carry a desiredmutation while others do not. In some cases, it is desirable to apply aselection pressure so as to enrich for bacteria carrying a desiredgenetic variation. Traditionally, selection for successful geneticvariants involved selection for or against some functionality impartedor abolished by the genetic variation, such as in the case of insertingantibiotic resistance gene or abolishing a metabolic activity capable ofconverting a non-lethal compound into a lethal metabolite. It is alsopossible to apply a selection pressure based on a polynucleotidesequence itself, such that only a desired genetic variation need beintroduced (e.g. without also requiring a selectable marker). In thiscase, the selection pressure can comprise cleaving genomes lacking thegenetic variation introduced to a target site, such that selection iseffectively directed against the reference sequence into which thegenetic variation is sought to be introduced. Typically, cleavage occurswithin 100 nucleotides of the target site (e.g. within 75, 50, 25, 10,or fewer nucleotides from the target site, including cleavage at orwithin the target site). Cleaving may be directed by a site-specificnuclease selected from the group consisting of a Zinc Finger nuclease, aCRISPR nuclease, a TALE nuclease (TALEN), and a meganuclease. Such aprocess is similar to processes for enhancing homologous recombinationat a target site, except that no template for homologous recombinationis provided. As a result, bacteria lacking the desired genetic variationare more likely to undergo cleavage that, left unrepaired, results incell death. Bacteria surviving selection may then be isolated for use inexposing to plants for assessing conferral of an improved trait.

A CRISPR nuclease may be used as the site-specific nuclease to directcleavage to a target site. An improved selection of mutated microbes canbe obtained by using Cas9 to kill non-mutated cells. Plants are theninoculated with the mutated microbes to re-confirm symbiosis and createevolutionary pressure to select for efficient symbionts. Microbes canthen be re-isolated from plant tissues. CRISPR nuclease systems employedfor selection against non-variants can employ similar elements to thosedescribed above with respect to introducing genetic variation, exceptthat no template for homologous recombination is provided. Cleavagedirected to the target site thus enhances death of affected cells.

Other options for specifically inducing cleavage at a target site areavailable, such as zinc finger nucleases, TALE nuclease (TALEN) systems,and meganuclease. Zinc-finger nucleases (ZFNs) are artificial DNAendonucleases generated by fusing a zinc finger DNA binding domain to aDNA cleavage domain. ZFNs can be engineered to target desired DNAsequences and this enables zinc-finger nucleases to cleave unique targetsequences. When introduced into a cell, ZFNs can be used to edit targetDNA in the cell (e.g., the cell's genome) by inducing double strandedbreaks. Transcription activator-like effector nucleases (TALENs) areartificial DNA endonucleases generated by fusing a TAL (Transcriptionactivator-like) effector DNA binding domain to a DNA cleavage domain.TALENS can be quickly engineered to bind practically any desired DNAsequence and when introduced into a cell, TALENs can be used to edittarget DNA in the cell (e.g., the cell's genome) by inducing doublestrand breaks. Meganucleases (homing endonuclease) areendodeoxyribonucleases characterized by a large recognition site(double-stranded DNA sequences of 12 to 40 base pairs. Meganucleases canbe used to replace, eliminate or modify sequences in a highly targetedway. By modifying their recognition sequence through proteinengineering, the targeted sequence can be changed. Meganucleases can beused to modify all genome types, whether bacterial, plant or animal andare commonly grouped into four families: the LAGLIDADG family (SEQ IDNO: 1), the GIY-YIG family, the His-Cyst box family and the HNH family.Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce,I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI,I-TevII and I-TevIII.

Genetic Variation—Methods of Identification

The microbes of the present disclosure may be identified by one or moregenetic modifications or alterations, which have been introduced intosaid microbe. One method by which said genetic modification oralteration can be identified is via reference to a SEQ ID NO thatcontains a portion of the microbe's genomic sequence that is sufficientto identify the genetic modification or alteration.

Further, in the case of microbes that have not had a geneticmodification or alteration (e.g. a wild type, WT) introduced into theirgenomes, the disclosure can utilize 16S nucleic acid sequences toidentify said microbes. A 16S nucleic acid sequence is an example of a“molecular marker” or “genetic marker,” which refers to an indicatorthat is used in methods for visualizing differences in characteristicsof nucleic acid sequences. Examples of other such indicators arerestriction fragment length polymorphism (RFLP) markers, amplifiedfragment length polymorphism (AFLP) markers, single nucleotidepolymorphisms (SNPs), insertion mutations, microsatellite markers(SSRs), sequence-characterized amplified regions (SCARs), cleavedamplified polymorphic sequence (CAPS) markers or isozyme markers orcombinations of the markers described herein which defines a specificgenetic and chromosomal location. Markers further include polynucleotidesequences encoding 16S or 18S rRNA, and internal transcribed spacer(ITS) sequences, which are sequences found between small-subunit andlarge-subunit rRNA genes that have proven to be especially useful inelucidating relationships or distinctions when compared against oneanother. Furthermore, the disclosure utilizes unique sequences found ingenes of interest (e.g. nifH, D, K, L, A, glnE, amtB, etc.) to identifymicrobes disclosed herein.

The primary structure of major rRNA subunit 16S comprise a particularcombination of conserved, variable, and hypervariable regions thatevolve at different rates and enable the resolution of both very ancientlineages such as domains, and more modern lineages such as genera. Thesecondary structure of the 16S subunit include approximately 50 heliceswhich result in base pairing of about 67% of the residues. These highlyconserved secondary structural features are of great functionalimportance and can be used to ensure positional homology in multiplesequence alignments and phylogenetic analysis. Over the previous fewdecades, the 16S rRNA gene has become the most sequenced taxonomicmarker and is the cornerstone for the current systematic classificationof bacteria and archaea (Yarza et al. 2014. Nature Rev. Micro.12:635-45).

Thus, in certain aspects, the disclosure provides for a sequence, whichshares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequencein Tables 23, 24, 30, 31, and 32.

Thus, in certain aspects, the disclosure provides for a microbe thatcomprises a sequence, which shares at least about 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NOs: 62-303. These sequences and theirassociated descriptions can be found in Tables 31 and 32.

In some aspects, the disclosure provides for a microbe that comprises a16S nucleic acid sequence, which shares at least about 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NOs: 85, 96, 111, 121, 122, 123, 124, 136,149, 157, 167, 261, 262, 269, 277-283. These sequences and theirassociated descriptions can be found in Table 32.

In some aspects, the disclosure provides for a microbe that comprises anucleic acid sequence, which shares at least about 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NOs: 86-95, 97-110, 112-120, 125-135,137-148, 150-156, 158-166, 168-176, 263-268, 270-274, 275, 276, 284-295.These sequences and their associated descriptions can be found in Table32.

In some aspects, the disclosure provides for a microbe that comprises anucleic acid sequence, which shares at least about 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NOs: 177-260, 296-303. These sequences andtheir associated descriptions can be found in Table 32.

In some aspects, the disclosure provides for a microbe that comprises,or primer that comprises, or probe that comprises, or non-nativejunction sequence that comprises, a nucleic acid sequence, which sharesat least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:304-424. These sequences and their associated descriptions can be foundin Table 30.

In some aspects, the disclosure provides for a microbe that comprises anon-native junction sequence that shares at least about 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NOs: 372-405. These sequences and theirassociated descriptions can be found in Table 30.

In some aspects, the disclosure provides for a microbe that comprises anamino acid sequence, which shares at least about 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NOs: 77, 78, 81, 82, or 83. These sequencesand their associated descriptions can be found in Table 31.

Genetic Variation—Methods of Detection: Primers, Probes, and Assays

The present disclosure teaches primers, probes, and assays that areuseful for detecting the microbes taught herein. In some aspects, thedisclosure provides for methods of detecting the WT parental strains. Inother aspects, the disclosure provides for methods of detecting thenon-intergeneric engineered microbes derived from the WT strains. Inaspects, the present disclosure provides methods of identifyingnon-intergeneric genetic alterations in a microbe.

In aspects, the genomic engineering methods of the present disclosurelead to the creation of non-natural nucleotide “junction” sequences inthe derived non-intergeneric microbes. These non-naturally occurringnucleotide junctions can be used as a type of diagnostic that isindicative of the presence of a particular genetic alteration in amicrobe taught herein.

The present techniques are able to detect these non-naturally occurringnucleotide junctions via the utilization of specialized quantitative PCRmethods, including uniquely designed primers and probes. In someaspects, the probes of the disclosure bind to the non-naturallyoccurring nucleotide junction sequences. In some aspects, traditionalPCR is utilized. In other aspects, real-time PCR is utilized. In someaspects, quantitative PCR (qPCR) is utilized.

Thus, the disclosure can cover the utilization of two common methods forthe detection of PCR products in real-time: (1) non-specific fluorescentdyes that intercalate with any double-stranded DNA, and (2)sequence-specific DNA probes consisting of oligonucleotides that arelabelled with a fluorescent reporter which permits detection only afterhybridization of the probe with its complementary sequence. In someaspects, only the non-naturally occurring nucleotide junction will beamplified via the taught primers, and consequently can be detected viaeither a non-specific dye, or via the utilization of a specifichybridization probe. In other aspects, the primers of the disclosure arechosen such that the primers flank either side of a junction sequence,such that if an amplification reaction occurs, then said junctionsequence is present.

Aspects of the disclosure involve non-naturally occurring nucleotidejunction sequence molecules per se, along with other nucleotidemolecules that are capable of binding to said non-naturally occurringnucleotide junction sequences under mild to stringent hybridizationconditions. In some aspects, the nucleotide molecules that are capableof binding to said non-naturally occurring nucleotide junction sequencesunder mild to stringent hybridization conditions are termed “nucleotideprobes.”

In aspects, genomic DNA can be extracted from samples and used toquantify the presence of microbes of the disclosure by using qPCR. Theprimers utilized in the qPCR reaction can be primers designed by PrimerBlast (www.ncbi.nlm.nih.gov/tools/primer-blast/) to amplify uniqueregions of the wild-type genome or unique regions of the engineerednon-intergeneric mutant strains. The qPCR reaction can be carried outusing the SYBR GreenER qPCR SuperMix Universal (Thermo Fisher P/N11762100) kit, using only forward and reverse amplification primers;alternatively, the Kapa Probe Force kit (Kapa Biosystems P/N KK4301) canbe used with amplification primers and a TaqMan probe containing a FAMdye label at the 5′ end, an internal ZEN quencher, and a minor groovebinder and fluorescent quencher at the 3′ end (Integrated DNATechnologies).

Certain primer, probe, and non-native junction sequences are listed inTable 30. qPCR reaction efficiency can be measured using a standardcurve generated from a known quantity of gDNA from the target genome.Data can be normalized to genome copies per g fresh weight using thetissue weight and extraction volume.

Quantitative polymerase chain reaction (qPCR) is a method ofquantifying, in real time, the amplification of one or more nucleic acidsequences. The real time quantification of the PCR assay permitsdetermination of the quantity of nucleic acids being generated by thePCR amplification steps by comparing the amplifying nucleic acids ofinterest and an appropriate control nucleic acid sequence, which may actas a calibration standard.

TaqMan probes are often utilized in qPCR assays that require anincreased specificity for quantifying target nucleic acid sequences.TaqMan probes comprise an oligonucleotide probe with a fluorophoreattached to the 5′ end and a quencher attached to the 3′ end of theprobe. When the TaqMan probes remain as is with the 5′ and 3′ ends ofthe probe in close contact with each other, the quencher preventsfluorescent signal transmission from the fluorophore. TaqMan probes aredesigned to anneal within a nucleic acid region amplified by a specificset of primers. As the Taq polymerase extends the primer and synthesizesthe nascent strand, the 5′ to 3′ exonuclease activity of the Taqpolymerase degrades the probe that annealed to the template. This probedegradation releases the fluorophore, thus breaking the close proximityto the quencher and allowing fluorescence of the fluorophore.Fluorescence detected in the qPCR assay is directly proportional to thefluorophore released and the amount of DNA template present in thereaction.

The features of qPCR allow the practitioner to eliminate thelabor-intensive post-amplification step of gel electrophoresispreparation, which is generally required for observation of theamplified products of traditional PCR assays. The benefits of qPCR overconventional PCR are considerable, and include increased speed, ease ofuse, reproducibility, and quantitative ability.

Improvement of Traits

Methods of the present disclosure may be employed to introduce orimprove one or more of a variety of desirable traits. Examples of traitsthat may introduced or improved include: root biomass, root length,height, shoot length, leaf number, water use efficiency, overallbiomass, yield, fruit size, grain size, photosynthesis rate, toleranceto drought, heat tolerance, salt tolerance, resistance to nematodestress, resistance to a fungal pathogen, resistance to a bacterialpathogen, resistance to a viral pathogen, level of a metabolite, andproteome expression. The desirable traits, including height, overallbiomass, root and/or shoot biomass, seed germination, seedling survival,photosynthetic efficiency, transpiration rate, seed/fruit number ormass, plant grain or fruit yield, leaf chlorophyll content,photosynthetic rate, root length, or any combination thereof, can beused to measure growth, and compared with the growth rate of referenceagricultural plants (e.g., plants without the improved traits) grownunder identical conditions.

A preferred trait to be introduced or improved is nitrogen fixation, asdescribed herein. In some cases, a plant resulting from the methodsdescribed herein exhibits a difference in the trait that is at leastabout 5% greater, for example at least about 5%, at least about 8%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 75%, at least about 80%, at least about80%, at least about 90%, or at least 100%, at least about 200%, at leastabout 300%, at least about 400% or greater than a reference agriculturalplant grown under the same conditions in the soil. In additionalexamples, a plant resulting from the methods described herein exhibits adifference in the trait that is at least about 5% greater, for exampleat least about 5%, at least about 8%, at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about75%, at least about 80%, at least about 80%, at least about 90%, or atleast 100%, at least about 200%, at least about 300%, at least about400% or greater than a reference agricultural plant grown under similarconditions in the soil.

The trait to be improved may be assessed under conditions including theapplication of one or more biotic or abiotic stressors. Examples ofstressors include abiotic stresses (such as heat stress, salt stress,drought stress, cold stress, and low nutrient stress) and bioticstresses (such as nematode stress, insect herbivory stress, fungalpathogen stress, bacterial pathogen stress, and viral pathogen stress).

The trait improved by methods and compositions of the present disclosuremay be nitrogen fixation, including in a plant not previously capable ofnitrogen fixation. In some cases, bacteria isolated according to amethod described herein produce 1% or more (e.g. 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, or more) of a plant's nitrogen, which mayrepresent an increase in nitrogen fixation capability of at least 2-fold(e.g. 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 50-fold, 100-fold, 1000-fold, or more) as compared to bacteriaisolated from the first plant before introducing any genetic variation.In some cases, the bacteria produce 5% or more of a plant's nitrogen.The desired level of nitrogen fixation may be achieved after repeatingthe steps of introducing genetic variation, exposure to a plurality ofplants, and isolating bacteria from plants with an improved trait one ormore times (e.g. 1, 2, 3, 4, 5, 10, 15, 25, or more times). In somecases, enhanced levels of nitrogen fixation are achieved in the presenceof fertilizer supplemented with glutamine, ammonia, or other chemicalsource of nitrogen. Methods for assessing degree of nitrogen fixationare known, examples of which are described herein.

Microbe breeding is a method to systematically identify and improve therole of species within the crop microbiome. The method comprises threesteps: 1) selection of candidate species by mapping plant-microbeinteractions and predicting regulatory networks linked to a particularphenotype, 2) pragmatic and predictable improvement of microbialphenotypes through intra-species crossing of regulatory networks andgene clusters, and 3) screening and selection of new microbial genotypesthat produce desired crop phenotypes. To systematically assess theimprovement of strains, a model is created that links colonizationdynamics of the microbial community to genetic activity by key species.The model is used to predict genetic targets for breeding and improvethe frequency of selecting improvements in microbiome-encoded traits ofagronomic relevance.

Measuring Nitrogen Delivered in an Agriculturally Relevant Field Context

In the field, the amount of nitrogen delivered can be determined by thefunction of colonization multiplied by the activity.

${{Nitrogen}\mspace{14mu}{delivered}} = {\int\limits_{{{Time}\mspace{14mu}\&}\mspace{14mu}{Space}}{{Colonization} \times {Activity}}}$

The above equation requires (1) the average colonization per unit ofplant tissue, and (2) the activity as either the amount of nitrogenfixed or the amount of ammonia excreted by each microbial cell. Toconvert to pounds of nitrogen per acre, corn growth physiology istracked over time, e.g., size of the plant and associated root systemthroughout the maturity stages.

The pounds of nitrogen delivered to a crop per acre-season can becalculated by the following equation:

Nitrogen delivered=∫Plant Tissue(t)×Colonization(t)×Activity(t)dt

The Plant Tissue(t) is the fresh weight of corn plant tissue over thegrowing time (t). Values for reasonably making the calculation aredescribed in detail in the publication entitled Roots, Growth andNutrient Uptake (Mengel. Dept. of Agronomy Pub. #AGRY-95-08 (Rev.May-95. p. 1-8).

The Colonization (t) is the amount of the microbes of interest foundwithin the plant tissue, per gram fresh weight of plant tissue, at anyparticular time, t, during the growing season. In the instance of only asingle timepoint available, the single timepoint is normalized as thepeak colonization rate over the season, and the colonization rate of theremaining timepoints are adjusted accordingly.

Activity(t) is the rate of which N is fixed by the microbes of interestper unit time, at any particular time, t, during the growing season. Inthe embodiments disclosed herein, this activity rate is approximated byin vitro acetylene reduction assay (ARA) in ARA media in the presence of5 mM glutamine or Ammonium excretion assay in ARA media in the presenceof 5 mM ammonium ions.

The Nitrogen delivered amount is then calculated by numericallyintegrating the above function. In cases where the values of thevariables described above are discretely measured at set timepoints, thevalues in between those timepoints are approximated by performing linearinterpolation.

Nitrogen Fixation

Described herein are methods of increasing nitrogen fixation in a plant,comprising exposing the plant to bacteria comprising one or more geneticvariations introduced into one or more genes regulating nitrogenfixation, wherein the bacteria produce 1% or more of nitrogen in theplant (e.g. 2%, 5%, 10%, or more), which may represent anitrogen-fixation capability of at least 2-fold as compared to the plantin the absence of the bacteria. The bacteria may produce the nitrogen inthe presence of fertilizer supplemented with glutamine, urea, nitratesor ammonia. Genetic variations can be any genetic variation describedherein, including examples provided above, in any number and anycombination. The genetic variation may be introduced into a geneselected from the group consisting of nifA, nifL, ntrB, ntrC, glutaminesynthetase, glnA, glnB, glnK, draT, amtB, glutaminase, glnD, glnE, nifJ,nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM,nifF, nifB, and nifQ. The genetic variation may be a mutation thatresults in one or more of: increased expression or activity of nifA orglutaminase; decreased expression or activity of nifL, ntrB, glutaminesynthetase, glnB, glnK, draT, amtB; decreased adenylyl-removing activityof GlnE; or decreased uridylyl-removing activity of GlnD. The geneticvariation introduced into one or more bacteria of the methods disclosedherein may be a knock-out mutation or it may abolish a regulatorysequence of a target gene, or it may comprise insertion of aheterologous regulatory sequence, for example, insertion of a regulatorysequence found within the genome of the same bacterial species or genus.The regulatory sequence can be chosen based on the expression level of agene in a bacterial culture or within plant tissue. The geneticvariation may be produced by chemical mutagenesis. The plants grown instep (c) may be exposed to biotic or abiotic stressors.

The amount of nitrogen fixation that occurs in the plants describedherein may be measured in several ways, for example by anacetylene-reduction (AR) assay. An acetylene-reduction assay can beperformed in vitro or in vivo. Evidence that a particular bacterium isproviding fixed nitrogen to a plant can include: 1) total plant Nsignificantly increases upon inoculation, preferably with a concomitantincrease in N concentration in the plant; 2) nitrogen deficiencysymptoms are relieved under N-limiting conditions upon inoculation(which should include an increase in dry matter); 3) N₂ fixation isdocumented through the use of an ¹⁵N approach (which can be isotopedilution experiments, ¹⁵N₂ reduction assays, or ¹⁵N natural abundanceassays); 4) fixed N is incorporated into a plant protein or metabolite;and 5) all of these effects are not be seen in non-inoculated plants orin plants inoculated with a mutant of the inoculum strain.

The wild-type nitrogen fixation regulatory cascade can be represented asa digital logic circuit where the inputs O₂ and NH₄ ⁺ pass through a NORgate, the output of which enters an AND gate in addition to ATP. In someembodiments, the methods disclosed herein disrupt the influence of NH₄ ⁺on this circuit, at multiple points in the regulatory cascade, so thatmicrobes can produce nitrogen even in fertilized fields. However, themethods disclosed herein also envision altering the impact of ATP or O₂on the circuitry, or replacing the circuitry with other regulatorycascades in the cell, or altering genetic circuits other than nitrogenfixation. Gene clusters can be re-engineered to generate functionalproducts under the control of a heterologous regulatory system. Byeliminating native regulatory elements outside of, and within, codingsequences of gene clusters, and replacing them with alternativeregulatory systems, the functional products of complex genetic operonsand other gene clusters can be controlled and/or moved to heterologouscells, including cells of different species other than the species fromwhich the native genes were derived. Once re-engineered, the syntheticgene clusters can be controlled by genetic circuits or other inducibleregulatory systems, thereby controlling the products' expression asdesired. The expression cassettes can be designed to act as logic gates,pulse generators, oscillators, switches, or memory devices. Thecontrolling expression cassette can be linked to a promoter such thatthe expression cassette functions as an environmental sensor, such as anoxygen, temperature, touch, osmotic stress, membrane stress, or redoxsensor.

As an example, the nifL, nifA, nifT, and nifX genes can be eliminatedfrom the nif gene cluster. Synthetic genes can be designed by codonrandomizing the DNA encoding each amino acid sequence. Codon selectionis performed, specifying that codon usage be as divergent as possiblefrom the codon usage in the native gene. Proposed sequences are scannedfor any undesired features, such as restriction enzyme recognitionsites, transposon recognition sites, repetitive sequences, sigma 54 andsigma 70 promoters, cryptic ribosome binding sites, and rho independentterminators. Synthetic ribosome binding sites are chosen to match thestrength of each corresponding native ribosome binding site, such as byconstructing a fluorescent reporter plasmid in which the 150 bpsurrounding a gene's start codon (from −60 to +90) is fused to afluorescent gene. This chimera can be expressed under control of thePtac promoter, and fluorescence measured via flow cytometry. To generatesynthetic ribosome binding sites, a library of reporter plasmids using150 bp (−60 to +90) of a synthetic expression cassette is generated.Briefly, a synthetic expression cassette can consist of a random DNAspacer, a degenerate sequence encoding an RBS library, and the codingsequence for each synthetic gene. Multiple clones are screened toidentify the synthetic ribosome binding site that best matched thenative ribosome binding site. Synthetic operons that consist of the samegenes as the native operons are thus constructed and tested forfunctional complementation. A further exemplary description of syntheticoperons is provided in US20140329326.

Bacterial Species

Microbes useful in the methods and compositions disclosed herein may beobtained from any source. In some cases, microbes may be bacteria,archaea, protozoa or fungi. The microbes of this disclosure may benitrogen fixing microbes, for example a nitrogen fixing bacteria,nitrogen fixing archaea, nitrogen fixing fungi, nitrogen fixing yeast,or nitrogen fixing protozoa. Microbes useful in the methods andcompositions disclosed herein may be spore-forming microbes, for examplespore forming bacteria. In some cases, bacteria useful in the methodsand compositions disclosed herein may be Gram positive bacteria or Gramnegative bacteria. In some cases, the bacteria may be an endosporeforming bacteria of the Firmicute phylum. In some cases, the bacteriamay be a diazotroph. In some cases, the bacteria may not be adiazotroph.

The methods and compositions of this disclosure may be used with anarchaea, such as, for example, Methanothermobacter thermoautotrophicus.

In some cases, bacteria which may be useful include, but are not limitedto, Agrobacterium radiobacter, Bacillus acidocaldarius, Bacillusacidoterrestris, Bacillus agri, Bacillus aizawai, Bacillus albolactis,Bacillus alcalophilus, Bacillus alvei, Bacillus aminoglucosidicus,Bacillus aminovorans, Bacillus amylolyticus (also known as Paenibacillusamylolyticus) Bacillus amyloliquefaciens, Bacillus aneurinolyticus,Bacillus atrophaeus, Bacillus azotoformans, Bacillus badius, Bacilluscereus (synonyms: Bacillus endorhythmos, Bacillus medusa), Bacilluschitinosporus, Bacillus circulars, Bacillus coagulans, Bacillusendoparasiticus Bacillus fastidiosus, Bacillus firmus, Bacilluskurstaki, Bacillus lacticola, Bacillus lactimorbus, Bacillus lactis,Bacillus laterosporus (also known as Brevibacillus laterosporus),Bacillus lautus, Bacillus lentimorbus, Bacillus lentus, Bacilluslicheniformis, Bacillus maroccanus, Bacillus megaterium, Bacillusmetiens, Bacillus mycoides, Bacillus natto, Bacillus nematocida,Bacillus nigrificans, Bacillus nigrum, Bacillus pantothenticus, Bacilluspopillae, Bacillus psychrosaccharolyticus, Bacillus pumilus, Bacillussiamensis, Bacillus smithii, Bacillus sphaericus, Bacillus subtilis,Bacillus thuringiensis, Bacillus uniflagellatus, Bradyrhizobiumjaponicum, Brevibacillus brevis Brevibacillus laterosporus (formerlyBacillus laterosporus), Chromobacterium subtsugae, Delftia acidovorans,Lactobacillus acidophilus, Lysobacter antibioticus, Lysobacterenzymogenes, Paenibacillus alvei, Paenibacillus polymyxa, Paenibacilluspopilliae (formerly Bacillus popilliae), Pantoea agglomerans, Pasteuriapenetrans (formerly Bacillus penetrans), Pasteuria usgae, Pectobacteriumcarotovorum (formerly Erwinia carotovora), Pseudomonas aeruginosa,Pseudomonas aureofaciens, Pseudomonas cepacia (formerly known asBurkholderia cepacia), Pseudomonas chlororaphis, Pseudomonasfluorescens, Pseudomonas proradix, Pseudomonas putida, Pseudomonassyringae, Serratia entomophila, Serratia marcescens, Streptomycescolombiensis, Streptomyces galbus, Streptomyces goshikiensis,Streptomyces griseoviridis, Streptomyces lavendulae, Streptomycesprasinus, Streptomyces saraceticus, Streptomyces venezuelae, Xanthomonascampestris, Xenorhabdus luminescens, Xenorhabdus nematophila,Rhodococcus globerulus AQ719 (NRRL Accession No. B-21663), Bacillus sp.AQ175 (ATCC Accession No. 55608), Bacillus sp. AQ 177 (ATCC AccessionNo. 55609), Bacillus sp. AQ178 (ATCC Accession No. 53522), andStreptomyces sp. strain NRRL Accession No. B-30145. In some cases thebacterium may be Azotobacter chroococcum, Methanosarcina barkeri,Klebsiella pneumoniae, Azotobacter vinelandii, Rhodobacter spharoides,Rhodobacter capsulatus, Rhodobacter palustris, Rhodosporillum rubrum,Rhizobium leguminosarum or Rhizobium etli.

In some cases, the bacterium may be a species of Clostridium, forexample Clostridium pasteurianum, Clostridium beijerinckii, Clostridiumperfringens, Clostridium tetani, Clostridium acetobutylicum.

In some cases, bacteria used with the methods and compositions of thepresent disclosure may be cyanobacteria. Examples of cyanobacterialgenuses include Anabaena (for example Anagaena sp. PCC7120), Nostoc (forexample Nostoc punctiforme), or Synechocystis (for example Synechocystissp. PCC6803).

In some cases, bacteria used with the methods and compositions of thepresent disclosure may belong to the phylum Chlorobi, for exampleChlorobium tepidum.

In some cases, microbes used with the methods and compositions of thepresent disclosure may comprise a gene homologous to a known NifH gene.Sequences of known NifH genes may be found in, for example, the Zehr labNifH database, (wwwzehr.pmc.ucsc.edu/nifH_Database_Public/, Apr. 4,2014), or the Buckley lab NifH database(www.css.cornell.edu/faculty/buckley/nifh, and Gaby, John Christian, andDaniel H. Buckley. “A comprehensive aligned nifH gene database: amultipurpose tool for studies of nitrogen-fixing bacteria.” Database2014 (2014): bau001). In some cases, microbes used with the methods andcompositions of the present disclosure may comprise a sequence whichencodes a polypeptide with at least 60%, 70%, 80%, 85%, 90%, 95%, 96%,96%, 98%, 99% or more than 99% sequence identity to a sequence from theZehr lab NifH database, (wwwzehr.pmc.ucsc.edu/nifH_Database_Public/,Apr. 4, 2014). In some cases, microbes used with the methods andcompositions of the present disclosure may comprise a sequence whichencodes a polypeptide with at least 60%, 70%, 80%, 85%, 90%, 95%, 96%,96%, 98%, 99% or more than 99% sequence identity to a sequence from theBuckley lab NifH database, (Gaby, John Christian, and Daniel H. Buckley.“A comprehensive aligned nifH gene database; a multipurpose tool forstudies of nitrogen-fixing bacteria.” Database 2014 (2014): bau00I).

Microbes useful in the methods and compositions disclosed herein can beobtained by extracting microbes from surfaces or tissues of nativeplants; grinding seeds to isolate microbes; planting seeds in diversesoil samples and recovering microbes from tissues; or inoculating plantswith exogenous microbes and determining which microbes appear in planttissues. Non-limiting examples of plant tissues include a seed,seedling, leaf, cutting, plant, bulb, tuber, root, and rhizomes. In somecases, bacteria are isolated from a seed. The parameters for processingsamples may be varied to isolate different types of associativemicrobes, such as rhizospheric, epiphytes, or endophytes. Bacteria mayalso be sourced from a repository, such as environmental straincollections, instead of initially isolating from a first plant. Themicrobes can be genotyped and phenotyped, via sequencing the genomes ofisolated microbes; profiling the composition of communities in planta;characterizing the transcriptomic functionality of communities orisolated microbes; or screening microbial features using selective orphenotypic media (e.g., nitrogen fixation or phosphate solubilizationphenotypes). Selected candidate strains or populations can be obtainedvia sequence data; phenotype data; plant data (e.g., genome, phenotype,and/or yield data); soil data (e.g., pH, N/P/K content, and/or bulk soilbiotic communities); or any combination of these.

The bacteria and methods of producing bacteria described herein mayapply to bacteria able to self-propagate efficiently on the leafsurface, root surface, or inside plant tissues without inducing adamaging plant defense reaction, or bacteria that are resistant to plantdefense responses. The bacteria described herein may be isolated byculturing a plant tissue extract or leaf surface wash in a medium withno added nitrogen. However, the bacteria may be unculturable, that is,not known to be culturable or difficult to culture using standardmethods known in the art. The bacteria described herein may be anendophyte or an epiphyte or a bacterium inhabiting the plant rhizosphere(rhizospheric bacteria). The bacteria obtained after repeating the stepsof introducing genetic variation, exposure to a plurality of plants, andisolating bacteria from plants with an improved trait one or more times(e.g. 1, 2, 3, 4, 5, 10, 15, 25, or more times) may be endophytic,epiphytic, or rhizospheric. Endophytes are organisms that enter theinterior of plants without causing disease symptoms or eliciting theformation of symbiotic structures, and are of agronomic interest becausethey can enhance plant growth and improve the nutrition of plants (e.g.,through nitrogen fixation). The bacteria can be a seed-borne endophyte.Seed-borne endophytes include bacteria associated with or derived fromthe seed of a grass or plant, such as a seed-borne bacterial endophytefound in mature, dry, undamaged (e.g., no cracks, visible fungalinfection, or prematurely germinated) seeds. The seed-borne bacterialendophyte can be associated with or derived from the surface of theseed; alternatively, or in addition, it can be associated with orderived from the interior seed compartment (e.g., of asurface-sterilized seed). In some cases, a seed-borne bacterialendophyte is capable of replicating within the plant tissue, forexample, the interior of the seed. Also, in some cases, the seed-bornebacterial endophyte is capable of surviving desiccation.

The bacterial isolated according to methods of the disclosure, or usedin methods or compositions of the disclosure, can comprise a pluralityof different bacterial taxa in combination. By way of example, thebacteria may include Proteobacteria (such as Pseudomonas, Enterobacter,Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea,Serratia, Rahnella, Azospirillum, Azorhizobium, Azotobacter, Duganella,Delftia, Bradyrhizobiun, Sinorhizobium and Halomonas), Firmicutes (suchas Bacillus, Paenibacillus, Lactobacillus, Mycoplasma, andAcetabacterium), and Actinobacteria (such as Streptomyces, Rhodacoccus,Microbacterium, and Curtobacterium). The bacteria used in methods andcompositions of this disclosure may include nitrogen fixing bacterialconsortia of two or more species. In some cases, one or more bacterialspecies of the bacterial consortia may be capable of fixing nitrogen. Insome cases, one or more species of the bacterial consortia mayfacilitate or enhance the ability of other bacteria to fix nitrogen. Thebacteria which fix nitrogen and the bacteria which enhance the abilityof other bacteria to fix nitrogen may be the same or different. In someexamples, a bacterial strain may be able to fix nitrogen when incombination with a different bacterial strain, or in a certain bacterialconsortia, but may be unable to fix nitrogen in a monoculture. Examplesof bacterial genuses which may be found in a nitrogen fixing bacterialconsortia include, but are not limited to, Herbaspirillum, Azospirillum,Enterobacter, and Bacillus.

Bacteria that can be produced by the methods disclosed herein includeAzotobacter sp., Bradyrhizobium sp., Klebsiella sp., and Sinorhizobiumsp. In some cases, the bacteria may be selected from the groupconsisting of: Azotobacter vinelandii, Bradyrhizobium japonicum,Klebsiella pneumoniae, and Sinorhizobium meliloti. In some cases, thebacteria may be of the genus Enterobacter or Rahnella. In some cases,the bacteria may be of the genus Frankia, or Clostridium. Examples ofbacteria of the genus Clostridium include, but are not limited to,Clostridium acetobutilicum, Clostridium pasteurianum, Clostridiumbeijerinckii, Clostridium perfringens, and Clostridium tetani. In somecases, the bacteria may be of the genus Paenibacillus, for examplePaenibacillus azotofixans, Paenibacillus borealis, Paenibacillus durus,Paenibacillus macerans, Paenibacillus polymyxa, Paenibacillus alvei,Paenibacillus amylolyticus, Paenibacillus campinasensis, Paenibacilluschibensis, Paenibacillus glucanolyticus, Paenibacillus illinoisensis,Paenibacillus larvae subsp. Larvae, Paenibacillus larvae subsp.Pulvifaciens, Paenibacillus lautus, Paenibacillus macerans,Paenibacillus macquariensis, Paenibacillus macquariensis, Paenibacilluspabuli, Paenibacillus peoriae, or Paenibacillus polymyxa.

In some examples, bacteria isolated according to methods of thedisclosure can be a member of one or more of the following taxa:Achromobacter, Acidithiobacillus, Acidovorax, Acidovoraz, Acinetobacter,Actinoplanes, Adlercreutzia, Aerococcus, Aeromonas, Afipia, Agromyces,Ancylobacter, Arthrobacter, Atopostipes, Azospirillum, Bacillus,Bdellovibrio, Beijerinckia, Bosea, Bradyrhizobium, Brevibacillus,Brevundimonas, Burkholderia, Candidatus Haloredivivus, Caulobacter,Cellulomonas, Cellvibrio, Chryseobacterium, Citrobacter, Clostridium,Coraliomargarita, Corynebacterium, Cupriavidus, Curtobacterium,Curvibacter, Deinococcus, Delftia, Desemzia, Devosia, Dokdonella,Dyella, Enhydrobacter, Enterobacter, Enterococcus, Erwinia, Escherichia,Escherichia/Shigella, Exiguobacterium, Ferroglobus, Filimonas,Finegoldia, Flavisolibacter, Flavobacterium, Frigoribacterium,Gluconacetobacter, Hafnia, Halobaculum, Halomonas, Halosimplex,Herbaspirillum, Hymenobacter, Klebsiella, Kocuria, Kosakonia,Lactobacillus, Leclercia, Lentzea, Luteibacter, Luteimonas, Massilia,Mesorhizobium, Methylobacterium, Microbacterium, Micrococcus,Microvirga, Mycobacterium, Neisseria, Nocardia, Oceanibaculum,Ochrobactrum, Okibacterium, Oligotropha, Oryzihumus, Oxalophagus,Paenibacillus, Panteoa, Pantoea, Pelomonas, Perlucidibaca, Plantibacter,Polynucleobacter, Propionibacterium, Propioniciclava, Pseudoclavibacter,Pseudomonas, Pseudonocardia, Pseudoxanthomonas, Psychrobacter, Rahnella,Ralstonia, Rheinheimera, Rhizobium, Rhodococcus, Rhodopseudomonas,Roseateles, Ruminococcus, Sebaldella, Sediminibacillus,Sediminibacterium, Serratia, Shigella, Shigella, Sinorhizobium,Sinosporangium, Sphingobacterium, Sphingomonas, Sphingopyxis,Sphingosinicella, Staphylococcus, 25 Stenotrophomonas,Strenotrophomonas, Streptococcus, Streptomyces, Stygiolobus,Sulfurisphaera, Tatumella, Tepidimonas, Thermomonas, Thiobacillus,Variovorax, WPS-2 genera incertae sedis, Xanthomonas, andZimmermannella.

In some cases, a bacterial species selected from at least one of thefollowing genera are utilized: Enterobacter, Klebsiella, Kosakonia, andRahnella. In some cases, a combination of bacterial species from thefollowing genera are utilized: Enterobacter, Klebsiella, Kosakonia, andRahnella. In some cases, the species utilized can be one or more of:Enterobacter sacchari, Klebsiella variicola, Kosakonia sacchari, andRahnella aquatilis.

In some cases, a Gram positive microbe may have a Molybdenum-Ironnitrogenase system comprising: nifH, nifD, nifK, nifB, nifE, nifN, nifX,hesA, nifV, nifW, nifU, nifS, and MN. In some cases, a Gram positivemicrobe may have a vanadium nitrogenase system comprising: vnfDG, vnfK,vnfE, vnfN, vupC, vupB, vupA, vnfV, vnfR1, vnfH, vnfR2, vnfA(transcriptional regulator). In some cases, a Gram positive microbe mayhave an iron-only nitrogenase system comprising: anfK, anfG, anfD, anfH,anfA (transcriptional regulator). In some cases a Gram positive microbemay have a nitrogenase system comprising glnB, and glnK (nitrogensignaling proteins). Some examples of enzymes involved in nitrogenmetabolism in Gram positive microbes include glnA (glutaminesynthetase), gdh (glutamate dehydrogenase), bdh (3-hydroxybutyratedehydrogenase), glutaminase, gltAB/gltB/gltS (glutamate synthase),asnA/asnB (aspartate-ammonia ligase/asparagine synthetase), andansA/ansZ (asparaginase). Some examples of proteins involved in nitrogentransport in Gram positive microbes include amtB (ammonium transporter),glnK (regulator of ammonium transport), glnPHQ/glnQHMP (ATP-dependentglutamine/glutamate transporters), glnT/alsT/yrbD/yflA (glutamine-likeproton symport transporters), and gltP/gltT/yhcl/nqt (glutamate-likeproton symport transporters).

Examples of Gram positive microbes which may be of particular interestinclude Paenibacillus polymixa, Paenibacillus riograndensis,Paenibacillus sp., Frankia sp., Heliobacterium sp., Heliobacteriumchlorum, Heliobacillus sp., Heliophilum sp., Heliorestis sp.,Clostridium acetobutylicum, Clostridium sp., Mycobacterium flaum,Mycobacterium sp., Arthrobacter sp., Agromyces sp., Corynebacteriumautitrophicum, Corynebacterium sp., Micromonspora sp., Propionibacteriasp., Streptomyces sp., and Microbacterium sp.

Some examples of genetic alterations which may be made in Gram positivemicrobes include: deleting glnR to remove negative regulation of BNF inthe presence of environmental nitrogen, inserting different promotersdirectly upstream of the nif cluster to eliminate regulation by GlnR inresponse to environmental nitrogen, mutating glnA to reduce the rate ofammonium assimilation by the GS-GOGAT pathway, deleting amtB to reduceuptake of ammonium from the media, mutating glnA so it is constitutivelyin the feedback-inhibited (FBI-GS) state, to reduce ammoniumassimilation by the GS-GOGAT pathway.

In some cases, glnR is the main regulator of N metabolism and fixationin Paenibacillus species. In some cases, the genome of a Paenibacillusspecies may not contain a gene to produce glnR. In some cases, thegenome of a Paenibacillus species may not contain a gene to produce glnEor glnD. In some cases, the genome of a Paenibacillus species maycontain a gene to produce glnB or glnK. For example, Paenibacillus sp.WLY78 doesn't contain a gene for glnB, or its homologs found in thearchaeon Methanococcus maripaludis, nifI1 and nifI2. In some cases, thegenomes of Paenibacillus species may be variable. For example,Paenibacillus polymixa E681 lacks glnK and gdh, has several nitrogencompound transporters, but only amtB appears to be controlled by GlnR.In another example, Paenibacillus sp. JDR2 has glnK, gdh and most othercentral nitrogen metabolism genes, has many fewer nitrogen compoundtransporters, but does have glnPHQ controlled by GlnR. Paenibacillusriograndensis SBR5 contains a standard glnRA operon, an fdx gene, a mainnif operon, a secondary nif operon, and an anf operon (encodingiron-only nitrogenase). Putative glnR/tnrA sites were found upstream ofeach of these operons. GlnR may regulate all of the above operons,except the anf operon. GlnR may bind to each of these regulatorysequences as a dimer.

Paenibacillus N-fixing strains may fall into two subgroups: Subgroup I,which contains only a minimal nif gene cluster and subgroup II, whichcontains a minimal cluster, plus an uncharacterized gene between nifXand hesA, and often other clusters duplicating some of the nif genes,such as nifH, nifHDK, nifBEN, or clusters encoding vanadium nitrogenase(vnf) or iron-only nitrogenase (anf) genes.

In some cases, the genome of a Paenibacillus species may not contain agene to produce glnB or glnK. In some cases, the genome of aPaenibacillus species may contain a minimal nif cluster with 9 genestranscribed from a sigma-70 promoter. In some cases, a Paenibacillus nifcluster may be negatively regulated by nitrogen or oxygen. In somecases, the genome of a Paenibacillus species may not contain a gene toproduce sigma-54. For example, Paenibacillus sp. WLY78 does not containa gene for sigma-54. In some cases, a nif cluster may be regulated byglnR, and/or TnrA. In some cases, activity of a nif cluster may bealtered by altering activity of glnR, and/or TnrA.

In Bacilli, glutamine synthetase (GS) is feedback-inhibited by highconcentrations of intracellular glutamine, causing a shift inconfirmation (referred to as FBI-GS). Nif clusters contain distinctbinding sites for the regulators GlnR and TnrA in several Bacillispecies. GlnR binds and represses gene expression in the presence ofexcess intracellular glutamine and AMP. A role of GlnR may be to preventthe influx and intracellular production of glutamine and ammonium underconditions of high nitrogen availability. TnrA may bind and/or activate(or repress) gene expression in the presence of limiting intracellularglutamine, and/or in the presence of FBI-GS. In some cases, the activityof a Bacilli nif cluster may be altered by altering the activity ofGlnR.

Feedback-inhibited glutamine synthetase (FBI-GS) may bind GlnR andstabilize binding of GlnR to recognition sequences. Several bacterialspecies have a GlnR/TnrA binding site upstream of the nif cluster.Altering the binding of FBI-GS and GlnR may alter the activity of thenif pathway.

Sources of Microbes

The bacteria (or any microbe according to the disclosure) may beobtained from any general terrestrial environment, including its soils,plants, fungi, animals (including invertebrates) and other biota,including the sediments, water and biota of lakes and rivers; from themarine environment, its biota and sediments (for example, sea water,marine muds, marine plants, marine invertebrates (for example, sponges),marine vertebrates (for example, fish)); the terrestrial and marinegeosphere (regolith and rock, for example, crushed subterranean rocks,sand and clays); the cryosphere and its meltwater; the atmosphere (forexample, filtered aerial dusts, cloud and rain droplets); urban,industrial and other man-made environments (for example, accumulatedorganic and mineral matter on concrete, roadside gutters, roof surfaces,and road surfaces).

The plants from which the bacteria (or any microbe according to thedisclosure) are obtained may be a plant having one or more desirabletraits, for example a plant that naturally grows in a particularenvironment or under certain conditions of interest. By way of example,a certain plant may naturally grow in sandy soil or sand of highsalinity, or under extreme temperatures, or with little water, or it maybe resistant to certain pests or disease present in the environment, andit may be desirable for a commercial crop to be grown in suchconditions, particularly if they are, for example, the only conditionsavailable in a particular geographic location. By way of furtherexample, the bacteria may be collected from commercial crops grown insuch environments, or more specifically from individual crop plants bestdisplaying a trait of interest amongst a crop grown in any specificenvironment: for example the fastest-growing plants amongst a crop grownin saline-limiting soils, or the least damaged plants in crops exposedto severe insect damage or disease epidemic, or plants having desiredquantities of certain metabolites and other compounds, including fibercontent, oil content, and the like, or plants displaying desirablecolors, taste or smell. The bacteria may be collected from a plant ofinterest or any material occurring in the environment of interest,including fungi and other animal and plant biota, soil, water,sediments, and other elements of the environment as referred topreviously.

The bacteria (or any microbe according to the disclosure) may beisolated from plant tissue. This isolation can occur from anyappropriate tissue in the plant, including for example root, stem andleaves, and plant reproductive tissues. By way of example, conventionalmethods for isolation from plants typically include the sterile excisionof the plant material of interest (e.g. root or stem lengths, leaves),surface sterilization with an appropriate solution (e.g. 2% sodiumhypochlorite), after which the plant material is placed on nutrientmedium for microbial growth. Alternatively, the surface-sterilized plantmaterial can be crushed in a sterile liquid (usually water) and theliquid suspension, including small pieces of the crushed plant materialspread over the surface of a suitable solid agar medium, or media, whichmay or may not be selective (e.g. contain only phytic acid as a sourceof phosphorus). This approach is especially useful for bacteria whichform isolated colonies and can be picked off individually to separateplates of nutrient medium, and further purified to a single species bywell-known methods. Alternatively, the plant root or foliage samples maynot be surface sterilized but only washed gently thus includingsurface-dwelling epiphytic microorganisms in the isolation process, orthe epiphytic microbes can be isolated separately, by imprinting andlifting off pieces of plant roots, stem or leaves onto the surface of anagar medium and then isolating individual colonies as above. Thisapproach is especially useful for bacteria, for example. Alternatively,the roots may be processed without washing off small quantities of soilattached to the roots, thus including microbes that colonize the plantrhizosphere. Otherwise, soil adhering to the roots can be removed,diluted and spread out onto agar of suitable selective and non-selectivemedia to isolate individual colonies of rhizospheric bacteria.

Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedures

The microbial deposits of the present disclosure were made under theprovisions of the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purpose of Patent Procedure(Budapest Treaty).

Applicants state that pursuant to 37 C.F.R. § 1.808 (a)(2) “allrestrictions imposed by the depositor on the availability to the publicof the deposited material will be irrevocably removed upon the grantingof the patent.” This statement is subject to paragraph (b) of thissection (i.e. 37 C.F.R. § 1.808 (b)).

The Enterobacter sacchari has now been reclassified as Kosakoniasacchari, the name for the organism may be used interchangeablythroughout the manuscript.

Many microbes of the present disclosure are derived from two wild-typestrains, as depicted in FIG. 6 and FIG. 7. Strain CI006 is a bacterialspecies previously classified in the genus Enterobacter (seeaforementioned reclassification into Kosakonia), and FIG. 6 identifiesthe lineage of the mutants that have been derived from CI006. StrainCI019 is a bacterial species classified in the genus Rahnella, and FIG.7 identifies the lineage of the mutants that have been derived fromCI019. With regard to FIG. 6 and FIG. 7, it is noted that strainscomprising CM in the name are mutants of the strains depictedimmediately to the left of said CM strain. The deposit information forthe CI006 Kosakonia wild type (WT) and CI019 Rahnella WT are found inthe below Table 1.

Some microorganisms described in this application were deposited on Jan.6, 2017 or Aug. 11, 2017 with the Bigelow National Center for MarineAlgae and Microbiota (NCMA), located at 60 Bigelow Drive, East Boothbay,Me. 04544, USA. As aforementioned, all deposits were made under theterms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure. TheBigelow National Center for Marine Algae and Microbiota accessionnumbers and dates of deposit for the aforementioned Budapest Treatydeposits are provided in Table 1.

Biologically pure cultures of Kosakonia sacchari (WT), Rahnellaaquatilis (WT), and a variant/remodeled Kosakonia sacchari strain weredeposited on Jan. 6, 2017 with the Bigelow National Center for MarineAlgae and Microbiota (NCMA), located at 60 Bigelow Drive, East Boothbay,Me. 04544, USA, and assigned NCMA Patent Deposit Designation numbers201701001, 201701003, and 201701002, respectively. The applicabledeposit information is found below in Table 1.

Biologically pure cultures of variant/remodeled Kosakonia saccharistrains were deposited on Aug. 11, 2017 with the Bigelow National Centerfor Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive,East Boothbay, Me. 04544, USA, and assigned NCMA Patent DepositDesignation numbers 201708004, 201708003, and 201708002, respectively.The applicable deposit information is found below in Table 1.

A biologically pure culture of Klebsiella variicola (WT) was depositedon Aug. 11, 2017 with the Bigelow National Center for Marine Algae andMicrobiota (NCMA), located at 60 Bigelow Drive, East Boothbay, Me.04544, USA, and assigned NCMA Patent Deposit Designation number201708001. Biologically pure cultures of two Klebsiella variicolavariants/remodeled strains were deposited on Dec. 20, 2017 with theBigelow National Center for Marine Algae and Microbiota (NCMA), locatedat 60 Bigelow Drive, East Boothbay, Me. 04544, USA, and assigned NCMAPatent Deposit Designation numbers 201712001 and 201712002,respectively. The applicable deposit information is found below in Table1.

TABLE 1 Microorganisms Deposited under the Budapest Treaty Pivot StrainDesignation (some strains have multiple Accession Date of Depositorydesignations) Taxonomy Number Deposit NCMA CI006, Kosakonia sacchari(WT) 201701001 Jan. 6, 2017 PBC6.1, 6 NCMA CI019, 19 Rahnella aquatilis(WT) 201701003 Jan. 6, 2017 NCMA CM029, 6-412 Kosakonia sacchari201701002 Jan. 6, 2017 NCMA 6-403 CM037 Kosakonia sacchari 201708004Aug. 11, 2017 NCMA 6-404, CM38, Kosakonia sacchari 201708003 Aug. 11,2017 PBC6.38 NCMA CM094, 6-881, Kosakonia sacchari 201708002 Aug. 11,2017 PBC6.94 NCMA CI137, 137, Klebsiella variicola (WT) 201708001 Aug.11, 2017 PB137 NCMA 137-1034 Klebsiella variicola 201712001 Dec. 20,2017 NCMA 137-1036 Klebsiella variicola 201712002 Dec. 20, 2017

Isolated and Biologically Pure Microorganisms

The present disclosure, in certain embodiments, provides isolated andbiologically pure microorganisms that have applications, inter alia, inagriculture. The disclosed microorganisms can be utilized in theirisolated and biologically pure states, as well as being formulated intocompositions (see below section for exemplary composition descriptions).Furthermore, the disclosure provides microbial compositions containingat least two members of the disclosed isolated and biologically puremicroorganisms, as well as methods of utilizing said microbialcompositions. Furthermore, the disclosure provides for methods ofmodulating nitrogen fixation in plants via the utilization of thedisclosed isolated and biologically pure microbes.

In some aspects, the isolated and biologically pure microorganisms ofthe disclosure are those from Table 1. In other aspects, the isolatedand biologically pure microorganisms of the disclosure are derived froma microorganism of Table 1. For example, a strain, child, mutant, orderivative, of a microorganism from Table 1 are provided herein. Thedisclosure contemplates all possible combinations of microbes listed inTable 1, said combinations sometimes forming a microbial consortia. Themicrobes from Table 1, either individually or in any combination, can becombined with any plant, active molecule (synthetic, organic, etc.),adjuvant, carrier, supplement, or biological, mentioned in thedisclosure.

In some aspects, the disclosure provides microbial compositionscomprising species as grouped in Tables 2-8. In some aspects, thesecompositions comprising various microbial species are termed a microbialconsortia or consortium.

With respect to Tables 2-8, the letters A through I represent anon-limiting selection of microorganisms of the present disclosure,defined as:

A=Microbe with accession number 201701001 identified in Table 1;

B=Microbe with accession number 201701003 identified in Table 1;

C=Microbe with accession number 201701002 identified in Table 1;

D=Microbe with accession number 201708004 identified in Table 1;

E=Microbe with accession number 201708003 identified in Table 1;

F=Microbe with accession number 201708002 identified in Table 1;

G=Microbe with accession number 201708001 identified in Table 1;

H=Microbe with accession number 201712001 identified in Table 1; and

I=Microbe with accession number 201712002 identified in Table 1.

TABLE 2 Eight and Nine Strain Compositions A, B, C, D, E, F, G, H A, B,C, D, E, F, G, I A, B, C, D, E, F, H, I A, B, C, D, E, G, H, I A, B, C,D, F, G, H, I A, B, C, E, F, G, H, I A, B, D, E, F, G, H, I A, C, D, E,F, G, H, I B, C, D, E, F, G, H, I A, B, C, D, E, F, G, H, I

TABLE 3 Seven Strain Compositions A, B, C, D, E, F, G A, B, C, D, E, F,H A, B, C, D, E, F, I A, B, C, D, E, G, H A, B, C, D, E, G, I A, B, C,D, E, H, I A, B, C, D, F, G, H A, B, C, D, F, G, I A, B, C, D, F, H, IA, B, C, D, G, H, I A, B, C, E, F, G, H A, B, C, E, F, G, I A, B, C, E,F, H, I A, B, C, E, G, H, I A, B, C, F, G, H, I A, B, D, E, F, G, H A,B, D, E, F, G, I A, B, D, E, F, H, I A, B, D, E, G, H, I A, B, D, F, G,H, I A, B, E, F, G, H, I A, C, D, E, F, G, H A, C, D, E, F, G, I A, C,D, E, F, H, I A, C, D, E, G, H, I A, C, D, F, G, H, I A, C, E, F, G, H,I A, D, E, F, G, H, I B, C, D, E, F, G, H B, C, D, E, F, G, I B, C, D,E, F, H, I B, C, D, E, G, H, I B, C, D, F, G, H, I B, C, E, F, G, H, IB, D, E, F, G, H, I C, D, E, F, G, H, I

TABLE 4 Six Strain Compositions A, B, C, D, E, F A, B, C, D, E, G A, B,C, D, E, H A, B, C, D, E, I A, B, C, D, F, G A, B, C, D, F, H A, B, C,D, F, I A, B, C, D, G, H A, B, C, D, G, I A, B, C, D, H, I A, B, C, E,F, G A, B, C, E, F, H A, B, C, E, F, I A, B, C, E, G, H A, B, C, E, G, IA, B, C, E, H, I A, B, C, F, G, H A, B, C, F, G, I A, B, C, F, H, I A,B, C, G, H, I A, B, D, E, F, G A, B, D, E, F, H A, B, D, E, F, I A, B,D, E, G, H A, B, D, E, G, I A, B, D, E, H, I A, B, D, F, G, H A, B, D,F, G, I D, E, F, G, H, I C, E, F, G, H, I A, B, D, F, H, I A, B, D, G,H, I A, B, E, F, G, H A, B, E, F, G, I A, B, E, F, H, I A, B, E, G, H, IA, B, F, G, H, I A, C, D, E, F, G A, C, D, E, F, H A, C, D, E, F, I A,C, D, E, G, H A, C, D, E, G, I A, C, D, E, H, I A, C, D, F, G, H A, C,D, F, G, I A, C, D, F, H, I A, C, D, G, H, I A, C, E, F, G, H A, C, E,F, G, I A, C, E, F, H, I A, C, E, G, H, I A, C, F, G, H, I A, D, E, F,G, H A, D, E, F, G, I A, D, E, F, H, I A, D, E, G, H, I A, D, F, G, H, IA, E, F, G, H, I B, C, D, E, F, G B, C, D, E, F, H B, C, D, E, F, I B,C, D, E, G, H B, C, D, E, G, I B, C, D, E, H, I B, C, D, F, G, H B, C,D, F, G, I B, C, D, F, H, I B, C, D, G, H, I B, C, E, F, G, H B, C, E,F, G, I B, C, E, F, H, I B, C, E, G, H, I B, C, F, G, H, I B, D, E, F,G, H B, D, E, F, G, I B, D, E, F, H, I B, D, E, G, H, I B, D, F, G, H, IB, E, F, G, H, I C, D, E, F, G, H C, D, E, F, G, I C, D, E, F, H, I C,D, E, G, H, I C, D, F, G, H, I

TABLE 5 Five Strain Compositions A, B, C, D, E A, B, C, D, F A, B, C, D,G A, B, C, D, H A, B, C, D, I A, B, C, E, F A, B, C, E, G A, B, C, E, HA, B, C, F, H A, B, C, F, G A, B, C, F, I A, B, C, G, H A, B, C, G, I A,B, C, H, I A, B, D, E, F A, B, D, E, G A, B, D, E, I A, B, D, F, G A, B,D, F, H A, B, D, F, I A, B, D, G, H A, B, D, G, I A, B, D, H, I A, B, E,F, G A, B, E, F, I A, B, E, G, H A, B, E, G, I A, B, E, H, I A, B, F, G,H A, B, F, G, I A, B, F, H, I A, B, G, H, I A, C, D, E, G A, C, D, E, HA, C, D, E, I A, C, D, F, G A, C, D, F, H A, C, D, F, I A, C, D, G, H A,C, D, G, I A, C, E, F, G A, C, E, F, H A, C, E, F, I A, C, E, G, H A, C,E, G, I A, C, E, H, I A, C, F, G, H A, C, F, G, I A, C, G, H, I A, D, E,F, G A, D, E, F, H A, D, E, F, I A, D, E, G, H A, D, E, G, I A, D, E, H,I A, D, F, G, H A, D, F, H, I A, D, G, H, I A, E, F, G, H A, E, F, G, IA, E, F, H, I A, E, G, H, I A, F, G, H, I B, C, D, E, F B, C, D, E, H B,C, D, E, I B, C, D, F, G B, C, D, F, H B, C, D, F, I B, C, D, G, H B, C,D, G, I B, C, D, H, I B, C, E, F, H B, C, E, F, I B, C, E, G, H B, C, E,G, I B, C, E, H, I B, C, F, G, H B, C, F, G, I B, C, F, H, I B, D, E, F,G B, D, E, F, H B, D, E, F, I B, D, E, G, H B, D, E, G, I B, D, E, H, IB, D, F, G, H B, D, F, G, I B, D, G, H, I B, E, F, G, H B, E, F, G, I B,E, F, H, I B, E, G, H, I B, F, G, H, I C, D, E, F, G C, D, E, F, H C, D,E, G, H C, D, E, G, I C, D, E, H, I C, D, F, G, H C, D, F, G, I C, D, F,H, I C, D, G, H, I C, E, F, G, H C, E, F, H, I C, E, G, H, I C, F, G, H,I D, E, F, G, H D, E, F, G, I D, E, F, H, I D, E, G, H, I D, F, G, H, IA, B, C, E, I A, B, D, E, H A, B, E, F, H A, C, D, E, F A, C, D, H, I A,C, F, H, I A, D, F, G, I B, C, D, E, G B, C, E, F, G B, C, G, H, I B, D,F, H, I C, D, E, F, I C, E, F, G, I E, F, G, H, I

TABLE 6 Four Strain Compositions A, B, C, D A, B, C, E A, B, C, F A, B,C, G A, B, C, H A, B, C, I A, B, D, E A, B, D, F D, G, H, I A, B, D, GA, B, D, H A, B, D, I A, B, E, F A, B, E, G A, B, E, H A, B, E, I A, B,F, G E, F, G, H A, B, F, H A, D, F, H A, D, F, I A, D, G, H A, D, G, IA, D, H, I A, E, F, G A, E, F, H E, F, G, I A, B, F, I A, B, G, H A, B,G, I A, B, H, I A, C, D, E A, C, D, F A, C, D, G A, C, D, H E, F, H, IA, C, D, I A, C, E, F A, C, E, G A, C, E, H A, C, E, I A, C, F, G A, C,F, H A, C, F, I E, G, H, I A, C, G, H A, C, G, I A, C, H, I A, D, E, FA, D, E, G A, D, E, H A, D, E, I A, D, F, G F, G, H, I A, E, F, I A, E,G, H A, E, G, I A, E, H, I A, F, G, H A, F, G, I A, F, H, I A, G, H, ID, E, F, H B, C, D, E B, C, D, F B, C, D, G B, C, D, H B, C, D, I B, C,E, F B, C, E, G B, C, E, H D, E, F, I B, C, E, I B, C, F, G B, C, F, HB, C, F, I B, C, G, H B, C, G, I B, C, H, I B, D, E, F D, E, G, H B, D,E, G B, D, E, H B, D, E, I B, D, F, G B, D, F, H B, D, F, I B, D, G, HB, D, G, I D, E, G, I B, D, H, I B, E, F, G B, E, F, H B, E, F, I B, E,G, H B, E, G, I B, E, H, I B, F, G, H D, E, H, I B, F, G, I B, F, H, IB, G, H, I C, D, E, F C, D, E, G C, D, E, H C, D, E, I C, D, F, G D, F,G, H C, D, F, H C, D, F, I C, D, G, H C, D, G, I C, D, H, I C, E, F, GC, E, F, H C, E, F, I D, F, G, I C, E, G, H C, E, G, I C, E, H, I C, F,G, H C, F, G, I C, F, H, I C, G, H, I D, E, F, G D, F, H, I

TABLE 7 Three Strain Compositions A, B, C A, B, D A, B, E A, B, F A, B,G A, B, H A, B, I A, C, D A, C, E G, H, I E, F, H A, C, F A, C, G A, C,H A, C, I A, D, E A, D, F A, D, G A, D, H A, D, I F, H, I E, F, G A, E,F A, E, G A, E, H A, E, I A, F, G A, F, H A, F, I A, G, H A, G, I F, G,I D, H, I A, H, I B, C, D B, C, E B, C, F B, C, G B, C, H B, C, I B, D,E B, D, F F, G, H D, G, I B, D, G B, D, H B, D, I B, E, F B, E, G B, E,H B, E, I B, F, G B, F, H E, H, I E, F, I B, F, I B, G, H B, G, I B, H,I C, D, E C, D, F C, D, G C, D, H C, D, I E, G, I D, G, H C, E, F C, E,G C, E, H C, E, I C, F, G C, F, H C, F, I C, G, H C, G, I E, G, H D, F,I C, H, I D, E, F D, E, G D, E, H D, E, I D, F, G D, F, H

TABLE 8 Two Strain Compositions A, B A, C A, D A, E A, F A, G A, H A, IB, C B, D B, E B, F B, G B, H B, I C, D C, E C, F C, G C, H C, I D, E D,F D, G D, H D, I E, F E, G E, H E, I F, G F, H F, I G, H G, I H, I

In some embodiments, microbial compositions may be selected from anymember group from Tables 2-8.

Agricultural Compositions

Compositions comprising bacteria or bacterial populations producedaccording to methods described herein and/or having characteristics asdescribed herein can be in the form of a liquid, a foam, or a dryproduct. Compositions comprising bacteria or bacterial populationsproduced according to methods described herein and/or havingcharacteristics as described herein may also be used to improve planttraits. In some examples, a composition comprising bacterial populationsmay be in the form of a dry powder, a slurry of powder and water, or aflowable seed treatment. The compositions comprising bacterialpopulations may be coated on a surface of a seed, and may be in liquidform.

The composition can be fabricated in bioreactors such as continuousstirred tank reactors, batch reactors, and on the farm. In someexamples, compositions can be stored in a container, such as a jug or inmini bulk. In some examples, compositions may be stored within an objectselected from the group consisting of a bottle, jar, ampule, package,vessel, bag, box, bin, envelope, carton, container, silo, shippingcontainer, truck bed, and/or case.

Compositions may also be used to improve plant traits. In some examples,one or more compositions may be coated onto a seed. In some examples,one or more compositions may be coated onto a seedling. In someexamples, one or more compositions may be coated onto a surface of aseed. In some examples, one or more compositions may be coated as alayer above a surface of a seed. In some examples, a composition that iscoated onto a seed may be in liquid form, in dry product form, in foamform, in a form of a slurry of powder and water, or in a flowable seedtreatment. In some examples, one or more compositions may be applied toa seed and/or seedling by spraying, immersing, coating, encapsulating,and/or dusting the seed and/or seedling with the one or morecompositions. In some examples, multiple bacteria or bacterialpopulations can be coated onto a seed and/or a seedling of the plant. Insome examples, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least nine, atleast ten, or more than ten bacteria of a bacterial combination can beselected from one of the following genera: Acidovorax, Agrobacterium,Bacillus, Burkholderia, Chryseobacterium, Curtobacterium, Enterobacter,Escherichia, Methylobacterium, Paenibacillus, Pantoea, Pseudomonas,Ralstonia, Saccharibacillus, Sphingomonas, and Stenotrophomonas.

In some examples, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least nine, atleast ten, or more than ten bacteria and bacterial populations of anendophytic combination are selected from one of the following families:Bacillaceae, Burkholderiaceae, Comamonadaceae, Enterobacteriaceae,Flavobacteriaceae, Methylobacteriaceae, Microbacteriaceae,Paenibacillileae, Pseudomonnaceae, Rhizobiaceae, Sphingomonadaceae,Xanthomonadaceae, Cladosporiaceae, Gnomoniaceae, Incertae sedis,Lasiosphaeriaceae, Netriaceae, and Pleosporaceae.

In some examples, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least night, atleast ten, or more than ten bacteria and bacterial populations of anendophytic combination are selected from one of the following families:Bacillaceae, Burkholderiaceae, Comamonadaceae, Enterobacteriaceae,Flavobacteriaceae, Methylobacteriaceae, Microbacteriaceae,Paenibacillileae, Pseudomonnaceae, Rhizobiaceae, Sphingomonadaceae,Xanthomonadaceae, Cladosporiaceae, Gnomoniaceae, Incertae sedis,Lasiosphaeriaceae, Netriaceae, Pleosporaceae.

Examples of compositions may include seed coatings for commerciallyimportant agricultural crops, for example, sorghum, canola, tomato,strawberry, barley, rice, maize, and wheat. Examples of compositions canalso include seed coatings for corn, soybean, canola, sorghum, potato,rice, vegetables, cereals, and oilseeds. Seeds as provided herein can begenetically modified organisms (GMO), non-GMO, organic, or conventional.In some examples, compositions may be sprayed on the plant aerial parts,or applied to the roots by inserting into furrows in which the plantseeds are planted, watering to the soil, or dipping the roots in asuspension of the composition. In some examples, compositions may bedehydrated in a suitable manner that maintains cell viability and theability to artificially inoculate and colonize host plants. Thebacterial species may be present in compositions at a concentration ofbetween 10⁸ to 10¹⁰ CFU/ml. In some examples, compositions may besupplemented with trace metal ions, such as molybdenum ions, iron ions,manganese ions, or combinations of these ions. The concentration of ionsin examples of compositions as described herein may between about 0.1 mMand about 50 mM. Some examples of compositions may also be formulatedwith a carrier, such as beta-glucan, carboxylmethyl cellulose (CMC),bacterial extracellular polymeric substance (EPS), sugar, animal milk,or other suitable carriers. In some examples, peat or planting materialscan be used as a carrier, or biopolymers in which a composition isentrapped in the biopolymer can be used as a carrier. The compositionscomprising the bacterial populations described herein can improve planttraits, such as promoting plant growth, maintaining high chlorophyllcontent in leaves, increasing fruit or seed numbers, and increasingfruit or seed unit weight.

The compositions comprising the bacterial populations described hereinmay be coated onto the surface of a seed. As such, compositionscomprising a seed coated with one or more bacteria described herein arealso contemplated. The seed coating can be formed by mixing thebacterial population with a porous, chemically inert granular carrier.Alternatively, the compositions may be inserted directly into thefurrows into which the seed is planted or sprayed onto the plant leavesor applied by dipping the roots into a suspension of the composition. Aneffective amount of the composition can be used to populate the sub-soilregion adjacent to the roots of the plant with viable bacterial growth,or populate the leaves of the plant with viable bacterial growth. Ingeneral, an effective amount is an amount sufficient to result in plantswith improved traits (e.g. a desired level of nitrogen fixation).

Bacterial compositions described herein can be formulated using anagriculturally acceptable carrier. The formulation useful for theseembodiments may include at least one member selected from the groupconsisting of a tackifier, a microbial stabilizer, a fungicide, anantibacterial agent, a preservative, a stabilizer, a surfactant, ananti-complex agent, an herbicide, a nematicide, an insecticide, a plantgrowth regulator, a fertilizer, a rodenticide, a desiccant, abactericide, a nutrient, and any combination thereof. In some examples,compositions may be shelf-stable. For example, any of the compositionsdescribed herein can include an agriculturally acceptable carrier (e.g.,one or more of a fertilizer such as a non-naturally occurringfertilizer, an adhesion agent such as a non-naturally occurring adhesionagent, and a pesticide such as a non-naturally occurring pesticide). Anon-naturally occurring adhesion agent can be, for example, a polymer,copolymer, or synthetic wax. For example, any of the coated seeds,seedlings, or plants described herein can contain such an agriculturallyacceptable carrier in the seed coating. In any of the compositions ormethods described herein, an agriculturally acceptable carrier can be orcan include a non-naturally occurring compound (e.g., a non-naturallyoccurring fertilizer, a non-naturally occurring adhesion agent such as apolymer, copolymer, or synthetic wax, or a non-naturally occurringpesticide). Non-limiting examples of agriculturally acceptable carriersare described below. Additional examples of agriculturally acceptablecarriers are known in the art.

In some cases, bacteria are mixed with an agriculturally acceptablecarrier. The carrier can be a solid carrier or liquid carrier, and invarious forms including microspheres, powders, emulsions and the like.The carrier may be any one or more of a number of carriers that confer avariety of properties, such as increased stability, wettability, ordispersability. Wetting agents such as natural or synthetic surfactants,which can be nonionic or ionic surfactants, or a combination thereof canbe included in the composition. Water-in-oil emulsions can also be usedto formulate a composition that includes the isolated bacteria (see, forexample, U.S. Pat. No. 7,485,451). Suitable formulations that may beprepared include wettable powders, granules, gels, agar strips orpellets, thickeners, and the like, microencapsulated particles, and thelike, liquids such as aqueous flowables, aqueous suspensions,water-in-oil emulsions, etc. The formulation may include grain or legumeproducts, for example, ground grain or beans, broth or flour derivedfrom grain or beans, starch, sugar, or oil.

In some embodiments, the agricultural carrier may be soil or a plantgrowth medium. Other agricultural carriers that may be used includewater, fertilizers, plant-based oils, humectants, or combinationsthereof. Alternatively, the agricultural carrier may be a solid, such asdiatomaceous earth, loam, silica, alginate, clay, bentonite,vermiculite, seed cases, other plant and animal products, orcombinations, including granules, pellets, or suspensions. Mixtures ofany of the aforementioned ingredients are also contemplated as carriers,such as but not limited to, pesta (flour and kaolin clay), agar orflour-based pellets in loam, sand, or clay, etc. Formulations mayinclude food sources for the bacteria, such as barley, rice, or otherbiological materials such as seed, plant parts, sugar cane bagasse,hulls or stalks from grain processing, ground plant material or woodfrom building site refuse, sawdust or small fibers from recycling ofpaper, fabric, or wood.

For example, a fertilizer can be used to help promote the growth orprovide nutrients to a seed, seedling, or plant. Non-limiting examplesof fertilizers include nitrogen, phosphorous, potassium, calcium,sulfur, magnesium, boron, chloride, manganese, iron, zinc, copper,molybdenum, and selenium (or a salt thereof). Additional examples offertilizers include one or more amino acids, salts, carbohydrates,vitamins, glucose, NaCl, yeast extract, NH₄H₂PO₄, (NH₄)₂SO₄, glycerol,valine, L-leucine, lactic acid, propionic acid, succinic acid, malicacid, citric acid, KH tartrate, xylose, lyxose, and lecithin. In oneembodiment, the formulation can include a tackifier or adherent(referred to as an adhesive agent) to help bind other active agents to asubstance (e.g., a surface of a seed). Such agents are useful forcombining bacteria with carriers that can contain other compounds (e.g.,control agents that are not biologic), to yield a coating composition.Such compositions help create coatings around the plant or seed tomaintain contact between the microbe and other agents with the plant orplant part. In one embodiment, adhesives are selected from the groupconsisting of: alginate, gums, starches, lecithins, formononetin,polyvinyl alcohol, alkali formononetinate, hesperetin, polyvinylacetate, cephalins, Gum Arabic, Xanthan Gum, Mineral Oil, PolyethyleneGlycol (PEG), Polyvinyl pyrrolidone (PVP), Arabino-galactan, MethylCellulose, PEG 400, Chitosan, Polyacrylamide, Polyacrylate,Polyacrylonitrile, Glycerol, Triethylene glycol, Vinyl Acetate, GellanGum, Polystyrene, Polyvinyl, Carboxymethyl cellulose, Gum Ghatti, andpolyoxyethylene-polyoxybutylene block copolymers.

In some embodiments, the adhesives can be, e.g. a wax such as carnaubawax, beeswax, Chinese wax, shellac wax, spermaceti wax, candelilla wax,castor wax, ouricury wax, and rice bran wax, a polysaccharide (e.g.,starch, dextrins, maltodextrins, alginate, and chitosans), a fat, oil, aprotein (e.g., gelatin and zeins), gum arables, and shellacs. Adhesiveagents can be non-naturally occurring compounds, e.g., polymers,copolymers, and waxes. For example, non-limiting examples of polymersthat can be used as an adhesive agent include: polyvinyl acetates,polyvinyl acetate copolymers, ethylene vinyl acetate (EVA) copolymers,polyvinyl alcohols, polyvinyl alcohol copolymers, celluloses (e.g.,ethylcelluloses, methylcelluloses, hydroxymethylcelluloses,hydroxypropylcelluloses, and carboxymethylcelluloses),polyvinylpyrolidones, vinyl chloride, vinylidene chloride copolymers,calcium lignosulfonates, acrylic copolymers, polyvinylacrylates,polyethylene oxide, acylamide polymers and copolymers, polyhydroxyethylacrylate, methylacrylamide monomers, and polychloroprene.

In some examples, one or more of the adhesion agents, anti-fungalagents, growth regulation agents, and pesticides (e.g., insecticide) arenon-naturally occurring compounds (e.g., in any combination). Additionalexamples of agriculturally acceptable carriers include dispersants(e.g., polyvinylpyrrolidone/vinyl acetate PVPIVA S-630), surfactants,binders, and filler agents.

The formulation can also contain a surfactant. Non-limiting examples ofsurfactants include nitrogen-surfactant blends such as Prefer 28(Cenex), Surf-N(US), Inhance (Brandt), P-28 (Wilfarm) and Patrol(Helena); esterified seed oils include Sun-It II (AmCy), MSO (UAP),Scoil (Agsco), Hasten (Wilfarm) and Mes-100 (Drexel); andorgano-silicone surfactants include Silwet L77 (UAP), Silikin (Terra),Dyne-Amic (Helena), Kinetic (Helena), Sylgard 309 (Wilbur-Ellis) andCentury (Precision). In one embodiment, the surfactant is present at aconcentration of between 0.01% v/v to 10% v/v. In another embodiment,the surfactant is present at a concentration of between 0.1% v/v to 1%v/v.

In certain cases, the formulation includes a microbial stabilizer. Suchan agent can include a desiccant, which can include any compound ormixture of compounds that can be classified as a desiccant regardless ofwhether the compound or compounds are used in such concentrations thatthey in fact have a desiccating effect on a liquid inoculant. Suchdesiccants are ideally compatible with the bacterial population used,and should promote the ability of the microbial population to surviveapplication on the seeds and to survive desiccation. Examples ofsuitable desiccants include one or more of trehalose, sucrose, glycerol,and Methylene glycol. Other suitable desiccants include, but are notlimited to, non-reducing sugars and sugar alcohols (e.g., mannitol orsorbitol). The amount of desiccant introduced into the formulation canrange from about 5% to about 50% by weight/volume, for example, betweenabout 10% to about 40%, between about 15% to about 35%, or between about20% to about 30%. In some cases, it is advantageous for the formulationto contain agents such as a fungicide, an antibacterial agent, anherbicide, a nematicide, an insecticide, a plant growth regulator, arodenticide, bactericide, or a nutrient. In some examples, agents mayinclude protectants that provide protection against seed surface-bornepathogens. In some examples, protectants may provide some level ofcontrol of soil-borne pathogens. In some examples, protectants may beeffective predominantly on a seed surface.

In some examples, a fungicide may include a compound or agent, whetherchemical or biological, that can inhibit the growth of a fungus or killa fungus. In some examples, a fungicide may include compounds that maybe fungistatic or fungicidal. In some examples, fungicide can be aprotectant, or agents that are effective predominantly on the seedsurface, providing protection against seed surface-borne pathogens andproviding some level of control of soil-borne pathogens. Non-limitingexamples of protectant fungicides include captan, maneb, thiram, orfludioxonil.

In some examples, fungicide can be a systemic fungicide, which can beabsorbed into the emerging seedling and inhibit or kill the fungusinside host plant tissues. Systemic fungicides used for seed treatmentinclude, but are not limited to the following: azoxystrobin, carboxin,mefenoxam, metalaxyl, thiabendazole, trifloxystrobin, and varioustriazole fungicides, including difenoconazole, ipconazole, tebuconazole,and triticonazole. Mefenoxam and metalaxyl are primarily used to targetthe water mold fungi Pythium and Phytophthora. Some fungicides arepreferred over others, depending on the plant species, either because ofsubtle differences in sensitivity of the pathogenic fungal species, orbecause of the differences in the fungicide distribution or sensitivityof the plants. In some examples, fungicide can be a biological controlagent, such as a bacterium or fungus. Such organisms may be parasitic tothe pathogenic fungi, or secrete toxins or other substances which cankill or otherwise prevent the growth of fungi. Any type of fungicide,particularly ones that are commonly used on plants, can be used as acontrol agent in a seed composition.

In some examples, the seed coating composition comprises a control agentthat has antibacterial properties. In one embodiment, the control agentwith antibacterial properties is selected from the compounds describedherein elsewhere. In another embodiment, the compound is Streptomycin,oxytetracycline, oxolinic acid, or gentamicin. Other examples ofantibacterial compounds which can be used as part of a seed coatingcomposition include those based on dichlorophene and benzylalcohol hemiformal (Proxel® from ICI or Acticide® RS from Thor Chemie and Kathon® MK25 from Rohm & Haas) and isothiazolinone derivatives such asalkylisothiazolinones and benzisothiazolinones (Acticide® MBS from ThorChemie).

In some examples, growth regulator is selected from the group consistingof: Abscisic acid, amidochlor, ancymidol, 6-benzylaminopurine,brassinolide, butralin, chlormequat (chlormequat chloride), cholinechloride, cyclanilide, daminozide, dikegulac, dimethipin,2,6-dimethylpuridine, ethephon, flumetralin, flurprimidol, fluthiacet,forchlorfenuron, gibberellic acid, inabenfide, indole-3-acetic acid,maleic hydrazide, mefluidide, mepiquat (mepiquat chloride),naphthaleneacetic acid, N-6-benzyladenine, paclobutrazol, prohexadionephosphorotrithioate, 2,3,5-tri-iodobenzoic acid, trinexapac-ethyl anduniconazole. Additional non-limiting examples of growth regulatorsinclude brassinosteroids, cytokinins (e.g., kinetin and zeatin), auxins(e.g., indolylacetic acid and indolylacetyl aspartate), flavonoids andisoflavanoids (e.g., formononetin and diosmetin), phytoaixins (e.g.,glyceolline), and phytoalexin-inducing oligosaccharides (e.g., pectin,chitin, chitosan, polygalacuronic acid, and oligogalacturonic acid), andgibellerins. Such agents are ideally compatible with the agriculturalseed or seedling onto which the formulation is applied (e.g., it shouldnot be deleterious to the growth or health of the plant). Furthermore,the agent is ideally one which does not cause safety concerns for human,animal or industrial use (e.g., no safety issues, or the compound issufficiently labile that the commodity plant product derived from theplant contains negligible amounts of the compound).

Some examples of nematode-antagonistic biocontrol agents include ARF18;30 Arthrobotrys spp.; Chaetomium spp.; Cylindrocarpon spp.; Exophiliaspp.; Fusarium spp.; Gliocladium spp.; Hirsutella spp.; Lecanicilliumspp.; Monacrosporium spp.; Myrothecium spp.; Neocosmospora spp.;Paecilomyces spp.; Pochonia spp.; Stagonospora spp.;vesicular-arbuscular mycorrhizal fungi, Burkholderia spp.; Pasteuriaspp., Brevibacillus spp.; Pseudomonas spp.; and Rhizobacteria.Particularly preferred nematode-antagonistic biocontrol agents includeARF18, Arthrobotrys oligospora, Arthrobotrys dactyloides, Chaetomiumglobosum, Cylindrocarpon heteronema, Exophilia jeanselmei, Exophiliapisciphila, Fusarium aspergilus, Fusarium solani, Gliocladiumcatenulatum, Gliocladium roseum, Gliocladium vixens, Hirsutellarhossiliensis, Hirsutella minnesotensis, Lecanicillium lecanii,Monacrosporium drechsleri, Monacrosporium gephyropagum, Myrotehciumverrucaria, Neocosmospora vasinfecta, Paecilomyces lilacinus, Pochoniachlamydosporia, Stagonospora heteroderae, Stagonospora phaseoli,vesicular-arbuscular mycorrhizal fungi, Burkholderia cepacia, Pasteuriapenetrans, Pasteuria thornei, Pasteuria nishizawae, Pasteuria ramosa,Pasteuria usage, Brevibacillus laterosporus strain G4, Pseudomonasfluorescens and Rhizobacteria.

Some examples of nutrients can be selected from the group consisting ofa nitrogen fertilizer including, but not limited to Urea, Ammoniumnitrate, Ammonium sulfate, Non-pressure nitrogen solutions, Aquaammonia, Anhydrous ammonia, Ammonium thiosulfate, Sulfur-coated urea,Urea-formaldehydes, IBDU, Polymer-coated urea, Calcium nitrate,Ureaform, and Methylene urea, phosphorous fertilizers such as Diammoniumphosphate, Monoammonium phosphate, Ammonium polyphosphate, Concentratedsuperphosphate and Triple superphosphate, and potassium fertilizers suchas Potassium chloride, Potassium sulfate, Potassium-magnesium sulfate,Potassium nitrate. Such compositions can exist as free salts or ionswithin the seed coat composition. Alternatively, nutrients/fertilizerscan be complexed or chelated to provide sustained release over time.

Some examples of rodenticides may include selected from the group ofsubstances consisting of 2-isovalerylindan-1,3-dione,4-(quinoxalin-2-ylamino) benzenesulfonamide, alpha-chlorohydrin,aluminum phosphide, antu, arsenous oxide, barium carbonate, bisthiosemi,brodifacoum, bromadiolone, bromethalin, calcium cyanide, chloralose,chlorophacinone, cholecalciferol, coumachlor, coumafuryl, coumatetralyl,crimidine, difenacoum, difethialone, diphacinone, ergocalciferol,flocoumafen, fluoroacetamide, flupropadine, flupropadine hydrochloride,hydrogen cyanide, iodomethane, lindane, magnesium phosphide, methylbromide, norbormide, phosacetim, phosphine, phosphorus, pindone,potassium arsenite, pyrinuron, scilliroside, sodium arsenite, sodiumcyanide, sodium fluoroacetate, strychnine, thallium sulfate, warfarinand zinc phosphide.

In the liquid form, for example, solutions or suspensions, bacterialpopulations can be mixed or suspended in water or in aqueous solutions.Suitable liquid diluents or carriers include water, aqueous solutions,petroleum distillates, or other liquid carriers.

Solid compositions can be prepared by dispersing the bacterialpopulations in and on an appropriately divided solid carrier, such aspeat, wheat, bran, vermiculite, clay, talc, bentonite, diatomaceousearth, fuller's earth, pasteurized soil, and the like. When suchformulations are used as wettable powders, biologically compatibledispersing agents such as non-ionic, anionic, amphoteric, or cationicdispersing and emulsifying agents can be used.

The solid carriers used upon formulation include, for example, mineralcarriers such as kaolin clay, pyrophyllite, bentonite, montmorillonite,diatomaceous earth, acid white soil, vermiculite, and pearlite, andinorganic salts such as ammonium sulfate, ammonium phosphate, ammoniumnitrate, urea, ammonium chloride, and calcium carbonate. Also, organicfine powders such as wheat flour, wheat bran, and rice bran may be used.The liquid carriers include vegetable oils such as soybean oil andcottonseed oil, glycerol, ethylene glycol, polyethylene glycol,propylene glycol, polypropylene glycol, etc.

Pests

Agricultural compositions of the disclosure, which may comprise anymicrobe taught herein, are sometimes combined with one or morepesticides.

The pesticides that are combined with the microbes of the disclosure maytarget any of the pests mentioned below.

“Pest” includes but is not limited to, insects, fungi, bacteria,nematodes, mites, ticks and the like. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera Orthroptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyLepidoptera and Coleoptera.

Those skilled in the art will recognize that not all compounds areequally effective against all pests. Compounds that may be combined withmicrobes of the disclosure may display activity against insect pests,which may include economically important agronomic, forest, greenhouse,nursery ornamentals, food and fiber, public and animal health, domesticand commercial structure, household and stored product pests.

As aforementioned, the agricultural compositions of the disclosure(which may comprise any microbe taught herein) are in embodimentscombined with one or more pesticides. These pesticides may be activeagainst any of the following pests:

Larvae of the order Lepidoptera include, but are not limited to,armyworms, cutworms, loopers and heliothines in the family NoctuidaeSpodoptera frugiperda J E Smith (fall armyworm); S. exigua Hubner (beetarmyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar);Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus(cabbage moth); Agrotis Ipsilon Hufnagel (black cutworm); A. orthogoniaMorrison (western cutworm); A. subterranea Fabricius (granulatecutworm); Alabama argillacea Hubner (cotton leaf worm); Trichoplusia niHubner (cabbage looper); Pseudoplusia includens Walker (soybean looper);Anticarsia gemmatalis Hubner (velvet bean caterpillar); Hypena scabraFabricius (green clover worm); Heliothis virescens Fabricius (tobaccobudworm); Pseudaletia umpuncta Haworth (armyworm); Athetis mindaraBarnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris(darksided cutworm); Earias insulana Boisduval (spiny bollworm); E.vittella Fabricius (spotted bollworm); Helicoverpa armigera Hubner(American bollworm); H. zea Boddie (corn earworm or cotton bollworm);Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialisGrote (citrus cutworm); borers, case bearers, webworms, coneworms, andskeletonizers from the family Pyralidae Ostrinia nubilalis Hubner(European corn borer); Amyelois transitella Walker (naval orangeworm);Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautellaWalker (almond moth); Chilo suppressalis Walker (rice stem borer); C.partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth);Crambus caliginosellus Clemens (corn root webworm); C. teterrellusZincken (bluegrass webworm); Cnaphalocrocis medinalis Guenee (rice leafroller); Desmia funeralis Hubner (grape leaffolder); Diaphania hyalinataLinnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraeagrandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius(surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestiaelutella Hubner (tobacco (cacao) moth); Galleria mellonella Linnaeus(greater wax moth); Herpetogramma licarsisalis Walker (sod webworm);Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellusZeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser waxmoth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalisWalker (tea tree web moth); Maruca testulalis Geyer (bean pod borer);Plodia interpunctella Hubner (Indian meal moth); Scirpophaga incertulasWalker (yellow stem borer); Udea rubigalis Guenee (celery leaftier); andleafrollers, budworms, seed worms and fruit worms in the familyTortricidae Acleris gloverana Walsingham (Western blackheaded budworm);A. variana Fernald (Eastern blackheaded budworm); Archips argyrospilaWalker (fruit tree leaf roller); A. rosana Linnaeus (European leafroller); and other Archips species, Adoxophyes orana Fischer vonRosslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham(banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C.pomonella Linnaeus (colding moth); Platynota flavedana Clemens(variegated leafroller); P. stultana Walsingham (omnivorous leafroller);Lobesia botrana Denis & Schiffermuller (European grape vine moth);Spilonota ocellana Denis & Schiffermuller (eyespotted bud moth);Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguellaHubner (vine moth); Bonagota salubricola Meyrick (Brazilian appleleafroller); Grapholita molesta Busck (oriental fruit moth); Suleimahelianthana Riley (sunflower bud moth); Argyrotaenia spp.; Choristoneuraspp.

Selected other agronomic pests in the order Lepidoptera include, but arenot limited to, Alsophila pometaria Harris (fall cankerworm); Anarsialineatella Zeller (peach twig borer); Anisota senatoria J. E. Smith(orange striped oakworm); Antheraea pernyi Guerin-Meneville (Chinese OakTussah Moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiellaBusck (cotton leaf perforator); Collas eurytheme Boisduval (alfalfacaterpillar); Datana integerrima Grote & Robinson (walnut caterpillar);Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomossubsignaria Hubner (elm spanworm); Erannis tiliaria Harris (lindenlooper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisinaamericana Guerin-Meneville (grapeleaf skeletonizer); Hemileuca oliviaeCockrell (range caterpillar); Hyphantria cunea Drury (fall web-worm);Keiferia lycopersicella Walsingham (tomato pinworm); Lambdinafiscellaria Hulst (Eastern hemlock looper); L. fiscellaria lugubrosaHulst (Western hemlock looper); Leucoma salicis Linnaeus (satin moth);Lymantria dispar Linnaeus (gypsy moth); Manduca quinquemaculata Haworth(five spotted hawk moth, tomato hornworm); M. sexta Haworth (tomatohomworm, tobacco hornworm); Operophtera brumata Linnaeus (winter moth);Paleacrita vernata Peck (spring cankerworm); Papilio cresphontes Cramer(giant swallowtail orange dog); Phryganidia californica Packard(California oakworm); Phyllocnistis citrella Stainton (citrusleafminer); Phyllonorycter blancardella Fabricius (spotted tentiformleafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapaeLinnaeus (small white butterfly); P. napi Linnaeus (green veined whitebutterfly); Platyptilia carduidactyla Riley (artichoke plume moth);Plutella xylostella Linnaeus (diamondback moth); Pectinophoragossypiella Saunders (pink bollworm); Pontia protodice Boisduval andLeconte (Southern cabbage-worm); Sabulodes aegrotata Guenee (onmivorouslooper); Schizura concinna J. E. Smith (red humped caterpillar);Sitotroga cerealella Olivier (Angoumois grain moth); Thaumetopoeapityocampa Schiffermuller (pine processionary caterpillar); Tineolabisselliella Hummel (webbing clothes moth); Tuta absoluta Meyrick(tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothissubflexa Guenee; Malacosoma spp. and Orgyia spp.; Ostrinia nubilalis(European corn borer); seed corn maggot; Agrotis ipsilon (blackcutworm).

Larvae and adults of the order Coleoptera including weevils from thefamilies Anthribidae, Bruchidae and Curculionidae (including, but notlimited to: Anthonomus grandis Boheman (boll weevil); Lissorhoptrusoryzophilus Kuschel (rice water weevil); Sitophilus granarius Linnaeus(granary weevil); S. oryzae Linnaeus (rice weevil); Hypera punctataFabricius (clover leaf weevil); Cylindrocopturus adspersus LeConte(sunflower stem weevil); Smicronyx fulvus LeConte (red sunflower seedweevil); S. sordidus LeConte (gray sunflower seed weevil); Sphenophorusmaidis Chittenden (maize billbug)); flea beetles, cucumber beetles,rootworms, leaf beetles, potato beetles and leafminers in the familyChrysomelidae (including, but not limited to: Leptinotarsa decemlineataSay (Colorado potato beetle); Diabrotica virgifera LeConte (western cornrootworm); D. barberi Smith and Lawrence (northern corn rootworm); D.undecimpunctata howardi Barber (southern corn rootworm); Chaetocnemapulicaria Melsheimer (corn flea beetle); Phyllotreta cruciferae Goeze(Crucifer flea beetle); Phyllotreta striolata (stripped flea beetle);Colaspis brunnea Fabricius (grape colaspis); Oulema melanopus Linnaeus(cereal leaf beetle); Zygogramma exclamationis Fabricius (sunflowerbeetle)); beetles from the family Coccinellidae (including, but notlimited to: Epilachna varivestis Mulsant (Mexican bean beetle)); chafersand other beetles from the family Scarabaeidae (including, but notlimited to: Popillia japonica Newman (Japanese beetle); Cyclocephalaborealis Arrow (northern masked chafer, white grub); C. immaculataOlivier (southern masked chafer, white grub); Rhizotrogus majalisRazoumowsky (European chafer); Phyllophaga crinita Burmeister (whitegrub); Ligyrus gibbosus De Geer (carrot beetle)); carpet beetles fromthe family Dermestidae; wireworms from the family Elateridae, Eleodesspp., Melanotus spp.; Conoderus spp.; Limonius spp.; Agriotes spp.;Ctenicera spp.; Aeolus spp.; bark beetles from the family Scolytidae andbeetles from the family Tenebrionidae; Cerotoma trifurcate (bean leafbeetle); and wireworm.

Adults and immatures of the order Diptera, including leafminers Agromyzaparvicornis Loew (corn blotch leafminer); midges (including, but notlimited to: Contarinia sorghicola Coquillett (sorghum midge); Mayetioladestructor Say (Hessian fly); Sitodiplosis mosellana Gehin (wheatmidge); Neolasioptera murtfeldtiana Felt, (sunflower seed midge)); fruitflies (Tephritidae), Oscinella frit Linnaeus (fruit flies); maggots(including, but not limited to: Delia platura Meigen (seedcorn maggot);D. coarctata Fallen (wheat bulb fly) and other Delia spp., Meromyzaamericana Fitch (wheat stem maggot); Musca domestica Linnaeus (houseflies); Fannia canicularis Linnaeus, F. femoralis Stein (lesser houseflies); Stomoxys calcitrans Linnaeus (stable flies)); face flies, hornflies, blow flies, Chrysomya spp.; Phormia spp. and other muscoid flypests, horse flies Tabanus spp.; bot flies Gastrophilus spp.; Oestrusspp.; cattle grubs Hypoderma spp.; deer flies Chrysops spp.; Melophagusovinus Linnaeus (keds) and other Brachycera, mosquitoes Aedes spp.;Anopheles spp.; Culex spp.; black flies Prosimulium spp.; Simulium spp.;biting midges, sand flies, sciarids, and other Nematocera.

Adults and nymphs of the orders Hemiptera and Homoptera such as, but notlimited to, adelgids from the family Adelgidae, plant bugs from thefamily Miridae, cicadas from the family Cicadidae, leafhoppers, Empoascaspp.; from the family Cicadellidae, planthoppers from the familiesCixiidae, Flatidae, Fulgoroidea, Issidae and Delphacidae, treehoppersfrom the family Membracidae, psyllids from the family Psyllidae,whiteflies from the family Aleyrodidae, aphids from the familyAphididae, phylloxera from the family Phylloxeridae, mealybugs from thefamily Pseudococcidae, scales from the families Asterolecanidae,Coccidae, Dactylopiidae, Diaspididae, Eriococcidae Ortheziidae,Phoenicococcidae and Margarodidae, lace bugs from the family Tingidae,stink bugs from the family Pentatomidae, cinch bugs, Blissus spp.; andother seed bugs from the family Lygaeidae, spittlebugs from the familyCercopidae squash bugs from the family Coreidae and red bugs and cottonstainers from the family Pyrrhocoridae.

Agronomically important members from the order Homoptera furtherinclude, but are not limited to: Acyrthisiphon pisum Harris (pea aphid);Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black beanaphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicisForbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecolaPatch (spirea aphid); Aulacorthum solani Kaltenbach (foxglove aphid);Chaetosiphon fragaefolii Cockerell (strawberry aphid); Diuraphis noxiaKurdjumov/Mordvilko (Russian wheat aphid); Dysaphis plantagineaPaaserini (rosy apple aphid); Eriosoma lanigerum Hausmann (woolly appleaphid); Brevicoryne brassicae Linnaeus (cabbage aphid); Hyalopteruspruni Geoffroy (mealy plum aphid); Lipaphis erysimi Kaltenbach (turnipaphid); Metopolophium dirrhodum Walker (cereal aphid); Macrosiphumeuphorbiae Thomas (potato aphid); Myzus persicae Sulzer (peach potatoaphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid);Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch(corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Schizaphisgraminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcaneaphid); Sitobion avenae Fabricius (English grain aphid); Therioaphismaculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer deFonscolombe (black citrus aphid) and T. citricida Kirkaldy (brown citrusaphid); Melanaphis sacchari (sugarcane aphid); Adelges spp. (adelgids);Phylloxera devastatrix Pergande (pecan phylloxera); Bemisia tabaciGennadius (tobacco whitefly, sweetpotato whitefly); B. argentifoliiBellows & Perring (silverleaf whitefly); Dialeurodes citri Ashmead(citrus whitefly); Trialeurodes abutiloneus (bandedwinged whitefly) andT. vaporariorum Westwood (greenhouse whitefly); Empoasca fabae Harris(potato leafhopper); Laodelphax striatellus Fallen (smaller brownplanthopper); Macrolestes quadrilineatus Forbes (aster leafhopper);Nephotettix cinticeps Uhler (green leafhopper); N. nigropictus Stal(rice leafhopper); Nilaparvata lugens Stal (brown planthopper);Peregrinus maidis Ashmead (corn planthopper); Sogatella furciferaHorvath (white backed planthopper); Sogatodes orizicola Muir (ricedelphacid); Typhlocyba pomaria McAtee (white apple leafhopper);Erythroneoura spp. (grape leafhoppers); Magicicada septendecim Linnaeus(periodical cicada); Icerya purchasi Maskell (cottony cushion scale);Quadraspidiotus perniciosus Comstock (San Jose scale); Planococcus citriRisso (citrus mealybug); Pseudococcus spp. (other mealybug complex);Cacopsylla pyricola Foerster (pear psylla); Trioza diospyri Ashmead(persimmon psylla).

Species from the order Hemiptera include, but are not limited to:Acrosternum hilare Say (green stink bug); Anasa tristis De Geer (squashbug); Blissus leucopterus Say (chinch bug); Corythuca gossypii Fabricius(cotton lace bug); Cyrtopeltis modesta Distant (tomato bug); Dysdercussuturellus Herrich-Schaffer (cotton stainer); Euschistus servus Say(brown stink bug); E. variolarius Palisot de Beauvais (one spotted stinkbug); Graptostethus spp. (complex of seed bugs); Leptoglossus corculusSay (leaf footed pine seed bug); Lygus lineolaris Palisot de Beauvais(tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug);L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius(European tarnished plant bug); Lygocoris pabulinus Linnaeus (commongreen capsid); Nezara viridula Linnaeus (southern green stink bug);Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas(large milk-weed bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper).

Hemiptera such as, Calocoris norvegicus Gmelin (strawberry bug); Orthopscampestris Linnaeus; Plesiocoris rugicollis Fallen (apple capsid);Cyrtopeltis modestus Distant (tomato bug); Cyrtopeltis notatus Distant(suckfly); Spanagonicus albofasciatus Reuter (whitemarked fleahopper);Diaphnocoris chlorionis Say (honeylocust plant bug); Labopidicola alliiKnight (onion plant bug); Pseudatomoscelis seriatus Reuter (cottonfleahopper); Adelphocoris rapidus Say (rapid plant bug); Poecilocapsuslineatus Fabricius (four lined plant bug); Nysius ericae Schilling(false chinch bug); Nysius raphanus Howard (false chinch bug); Nezaraviridula Linnaeus (Southern green stink bug); Eurygaster spp.; Coreidaespp.; Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.; Reduviidaespp. and Cimicidae spp.

Adults and larvae of the order Acari (mites) such as Aceria tosichellaKeifer (wheat curl mite); Petrobia latens Muller (brown wheat mite);spider mites and red mites in the family Tetranychidae, Panonychus ulmiKoch (European red mite); Tetranychus urticae Koch (two spotted spidermite); (T. mcdanieli McGregor (McDaniel mite); T. cinnabarinus Boisduval(carmine spider mite); T. turkestani Ugarov & Nikolski (strawberryspider mite); flat mites in the family Tenuipalpidae, Brevipalpus lewisiMcGregor (citrus flat mite); rust and bud mites in the familyEriophyidae and other foliar feeding mites and mites important in humanand animal health, i.e., dust mites in the family Epidermoptidae,follicle mites in the family Demodicidae, grain mites in the familyGlycyphagidae, ticks in the order Ixodidae. Ixodes scapularis Say (deertick); I. holocyclus Neumann (Australian paralysis tick); Dermacentorvariabilis Say (American dog tick); Amblyomma americanum Linnaeus (lonestar tick) and scab and itch mites in the families Psoroptidae,Pyemotidae and Sarcoptidae.

Insect pests of the order Thysanura, such as Lepisma saccharina Linnaeus(silverfish); Thermobia domestica Packard (firebrat).

Additional arthropod pests include: spiders in the order Araneae such asLoxosceles reclusa Gertsch and Mulaik (brown recluse spider) and theLatrodectus mactans Fabricius (black widow spider) and centipedes in theorder Scutigeromorpha such as Scutigera coleoptrata Linnaeus (housecentipede).

Superfamily of stink bugs and other related insects including but notlimited to species belonging to the family Pentatomidae (Nezaraviridula, Halyomorpha halys, Piezodorus guildini, Euschistus servus,Acrosternum hilare, Euschistus heros, Euschistus tristigmus, Acrosternumhilare, Dichelops furcatus, Dichelops melacanthus, and Bagrada hilaris(Bagrada Bug)), the family Plataspidae (Megacopta cribraria-Beanplataspid) and the family Cydnidae (Scaptocoris castanea-Root stink bug)and Lepidoptera species including but not limited to: diamond-back moth,e.g., Helicoverpa zea Boddie; soybean looper, e.g., Pseudoplusiaincludens Walker and velvet bean caterpillar e.g., Anticarsia gemmatalisHubner.

Nematodes include parasitic nematodes such as root-knot, cyst and lesionnematodes, including Heterodera spp., Meloidogyne spp. and Globoderaspp.; particularly members of the cyst nematodes, including, but notlimited to, Heterodera glycines (soybean cyst nematode); Heteroderaschachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode)and Globodera rostochiensis and Globodera pailida (potato cystnematodes). Lesion nematodes include Pratylenchus spp.

Pesticidal Compositions Comprising a Pesticide and Microbe of theDisclosure

As aforementioned, agricultural compositions of the disclosure, whichmay comprise any microbe taught herein, are sometimes combined with oneor more pesticides. Pesticides can include herbicides, insecticides,fungicides, nematicides, etc.

In some embodiments, the pesticides/microbial combinations can beapplied in the form of compositions and can be applied to the crop areaor plant to be treated, simultaneously or in succession, with othercompounds. These compounds can be fertilizers, weed killers,cryoprotectants, surfactants, detergents, pesticidal soaps, dormantoils, polymers, and/or time release or biodegradable carrierformulations that permit long term dosing of a target area following asingle application of the formulation. They can also be selectiveherbicides, chemical insecticides, virucides, microbicides, amoebicides,pesticides, fungicides, bacteriocides, nematicides, molluscicides ormixtures of several of these preparations, if desired, together withfurther agriculturally acceptable carriers, surfactants or applicationpromoting adjuvants customarily employed in the art of formulation.Suitable carriers (i.e. agriculturally acceptable carriers) andadjuvants can be solid or liquid and correspond to the substancesordinarily employed in formulation technology, e.g. natural orregenerated mineral substances, solvents, dispersants, wetting agents,sticking agents, tackifiers, binders or fertilizers. Likewise, theformulations may be prepared into edible baits or fashioned into pesttraps to permit feeding or ingestion by a target pest of the pesticidalformulation.

Exemplary chemical compositions, which may be combined with the microbesof the disclosure, include:

Fruits/Vegetables Herbicides: Atrazine, Bromacil, Diuron, Glyphosate,Linuron, Metribuzin, Simazine, Trifluralin, Fluazifop, Glufosinate, Halosulfuron Gowan, Paraquat, Propyzamide, Sethoxydim, Butafenacil,Halosulfuron, Indaziflam; Fruits/Vegetables Insecticides: Aldicarb,Bacillus thuringiensis, Carbaryl, Carbofuran, Chlorpyrifos,Cypermethrin, Deltamethrin, Diazinon, Malathion, Abamectin,Cyfluthrin/betacyfluthrin, Esfenvalerate, Lambda-cyhalothrin,Acequinocyl, Bifenazate, Methoxyfenozide, Novaluron, Chromafenozide,Thiacloprid, Dinotefuran, FluaCrypyrim, Tolfenpyrad, Clothianidin,Spirodiclofen, Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr,Cyazypyr, Spinoteram, Triflumuron, Spirotetramat, Imidacloprid,Flubendiamide, Thiodicarb, Metaflumizone, Sulfoxaflor, Cyflumetofen,Cyanopyrafen, Imidacloprid, Clothianidin, Thiamethoxam, Spinotoram,Thiodicarb, Flonicamid, Methiocarb, Emamectin benzoate, Indoxacarb,Forthiazate, Fenamiphos, Cadusaphos, Pyriproxifen, Fenbutatin oxide,Hexthiazox, Methomyl,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on;Fruits Vegetables Fungicides: Carbendazim, Chlorothalonil, EBDCs,Sulphur, Thiophanate-methyl, Azoxystrobin, Cymoxanil, Fluazinam,Fosetyl, Iprodione, Kresoxim-methyl, Metalaxyl/mefenoxam,Trifloxystrobin, Ethaboxam, Iprovalicarb, Trifloxystrobin, Fenhexamid,Oxpoconazole fumarate, Cyazofamid, Fenamidone, Zoxamide, Picoxystrobin,Pyraclostrobin, Cyflufenamid, Boscalid;

Cereals Herbicides: Isoproturon, Bromoxynil, Ioxynil, Phenoxies,Chlorsulfuron, Clodinafop, Diclofop, Diflufenican, Fenoxaprop,Florasulam, Fluoroxypyr, Metsulfuron, Triasulfuron, Flucarbazone,Iodosulfuron, Propoxycarbazone, Picolin-afen, Mesosulfuron,Beflubutamid, Pinoxaden, Amidosulfuron, Thifensulfuron Methyl,Tribenuron, Flupyrsulfuron, Sulfosulfuron, Pyrasulfotole, Pyroxsulam,Flufenacet, Tralkoxydim, Pyroxasulfon; Cereals Fungicides: Carbendazim,Chlorothalonil, Azoxystrobin, Cyproconazole, Cyprodinil, Fenpropimorph,Epoxiconazole, Kresoxim-methyl, Quinoxyfen, Tebuconazole,Trifloxystrobin, Simeconazole, Picoxystrobin, Pyraclostrobin,Dimoxystrobin, Prothioconazole, Fluoxastrobin; Cereals Insecticides:Dimethoate, Lambda-cyhalothrin, Deltamethrin, alpha-Cypermethrin,β-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Clorphyriphos, Metamidophos,Oxidemethon methyl, Pirimicarb, Methiocarb;

Maize Herbicides: Atrazine, Alachlor, Bromoxynil, Acetochlor, Dicamba,Clopyralid, S-Dimethenamid, Glufosinate, Glyphosate, Isoxaflutole,S-Metolachlor, Mesotrione, Nicosulfuron, Primisulfuron, Rimsulfuron,Sulcotrione, Foramsulfuron, Topramezone, Tembotrione, Saflufenacil,Thiencarbazone, Flufenacet, Pyroxasulfon; Maize Insecticides:Carbofuran, Chlorpyrifos, Bifenthrin, Fipronil, Imidacloprid,Lambda-Cyhalothrin, Tefluthrin, Terbufos, Thiamethoxam, Clothianidin,Spiromesifen, Flubendiamide, Triflumuron, Rynaxypyr, Deltamethrin,Thiodicarb, β-Cyfluthrin, Cypermethrin, Bifenthrin, Lufenuron,Triflumoron, Tefluthrin, Tebupirim-phos, Ethiprole, Cyazypyr,Thiacloprid, Acetamiprid, Dinetofuran, Avermectin, Methiocarb,Spirodiclofen, Spirotetramat; Maize Fungicides: Fenitropan, Thiram,Prothioconazole, Tebuconazole, Trifloxystrobin;

Rice Herbicides: Butachlor, Propanil, Azimsulfuron, Bensulfuron,Cyhalo-fop, Daimuron, Fentrazamide, Imazosulfuron, Mefenacet,Oxaziclomefone, Pyrazosulfuron, Pyributicarb, Quinclorac, Thiobencarb,Indanofan, Flufenacet, Fentrazamide, Halosulfuron, Oxaziclomefone,Benzobicyclon, Pyriftalid, Penoxsulam, Bispyribac, Oxadiargyl,Ethoxysulfuron, Pretilachlor, Mesotrione, Tefuryltrione, Oxadiazone,Fenoxaprop, Pyrimisulfan; Rice Insecticides: Diazinon, Fenitro-thion,Fenobucarb, Monocrotophos, Benfuracarb, Buprofezin, Dinotefuran,Fipronil, Imidacloprid, Isoprocarb, Thiacloprid, Chromafenozide,Thiacloprid, Dinotefuran, Clothianidin, Ethiprole, Flubendiamide,Rynaxypyr, Deltamethrin, Acetamiprid, Thiamethoxam, Cyazypyr, Spinosad,Spinotoram, Emamectin-Benzoate, Cypermethrin, Chlorpyriphos, Cartap,Methamidophos, Etofen-prox, Triazophos,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Carbofuran, Benfuracarb; Rice Fungicides: Thiophanate-methyl,Azoxystrobin, Carpropamid, Edifenphos, Ferimzone, Iprobenfos,Isoprothiolane, Pencycuron, Probenazole, Pyroquilon, Tricyclazole,Trifloxystrobin, Diclocymet, Fenoxanil, Simeconazole, Tiadinil;

Cotton Herbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn,Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate,Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron,Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; CottonInsecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin,Deltamethrin, Malathion, Monocrotophos, Abamectin, Acetamiprid,Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin,Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl,Flonicamid, Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin,Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran,Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin,4-[[(6-Chlorpyridin-3-yl) methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen,Sulfoxaflor, Profenophos, Thriazophos, Endosulfan; Cotton Fungicides:Etridiazole, Metalaxyl, Quintozene;

Soybean Herbicides: Alachlor, Bentazone, Trifluralin, Chlorimuron-Ethyl,Cloransulam-Methyl, Fenoxaprop, Fomesafen, Flu-azifop, Glyphosate,Imazamox, Imazaquin, Imazethapyr, (S-)Metolachlor, Metribuzin,Pendimethalin, Tepraloxydim, Glufosinate; Soybean Insecticides:Lambda-cyhalothrin, Methomyl, Parathion, Thiocarb, Imidacloprid,Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran,Flubendiamide, Rynaxypyr, Cyazypyr, Spinosad, Spinotoram,Emamectin-Benzoate, Fipronil, Ethiprole, Deltamethrin, β-Cyfluthrin,gamma and lambda Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Spirotetramat,Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb, beta-Cyfluthrin;Soybean Fungicides: Azoxystrobin, Cyproconazole, Epoxiconazole,Flutriafol, Pyraclostrobin, Tebuconazole, Trifloxystrobin,Prothioconazole, Tetraconazole;

Sugarbeet Herbicides: Chloridazon, Desmedipham, Ethofumesate,Phenmedipham, Triallate, Clopyralid, Fluazifop, Lenacil, Metamitron,Quinmerac, Cycloxydim, Triflusulfuron, Tepral-oxydim, Quizalofop;Sugarbeet Insecticides: Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Deltamethrin, β-Cyfluthrin,gamma/lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluor-ethyl)amino]furan-2(5H)-on,Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran;

Canola Herbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate,Glyphosate, Metazachlor, Trifluralin Ethametsulfuron, Quinmerac,Quizalofop, Clethodim, Tepraloxydim; Canola Fungicides: Azoxystrobin,Carbendazim, Fludioxonil, Iprodione, Prochloraz, Vinclozolin; CanolaInsecticides: Carbofuran organophos-phates, Pyrethroids, Thiacloprid,Deltamethrin, Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid,Dineto-furan, β-Cyfluthrin, gamma and lambda Cyhalothrin,tau-Fluvaleriate, Ethiprole, Spinosad, Spinotoram, Flubendiamide,Rynaxypyr, Cyazypyr,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on.

Insecticidal Compositions Comprising an Insecticide and Microbe of theDisclosure

As aforementioned, agricultural compositions of the disclosure, whichmay comprise any microbe taught herein, are sometimes combined with oneor more insecticides.

In some embodiments, insecticidal compositions may be included in thecompositions set forth herein, and can be applied to a plant(s) or apart(s) thereof simultaneously or in succession, with other compounds.Insecticides include ammonium carbonate, aqueous potassium silicate,boric acid, copper sulfate, elemental sulfur, lime sulfur, sucroseoctanoate esters,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,abamectin, notenone, fenazaquin, fenpyroximate, pyridaben, pyrimedifen,tebufenpyrad, tolfenpyrad, acephate, emamectin benzoate, lepimectin,milbemectin, hdroprene, kinoprene, methoprene, fenoxycarb, pyriproxyfen,methryl bromide and other alkyl halides, fulfuryl fluoride,chloropicrin, borax, disodium octaborate, sodium borate, sodiummetaborate, tartar emetic, dazomet, metam, pymetrozine, pyrifluquinazon,flofentezine, diflovidazin, hexythiazox, bifenazate, thiamethoxam,imidacloprid, fenpyroximate, azadirachtin, permethrin, esfenvalerate,acetamiprid, bifenthrin, indoxacarb, azadirachtin, pyrethrin,imidacloprid, beta-cyfluthrin, sulfotep, tebupirimfos, temephos,terbufos, tetrachlorvinphos, thiometon, triazophos, alanycarb, aldicarb,bendiocarb, benfluracarb, butocarboxim, butoxycarboxim, carbaryl,carbofuran, carbosulfan, ethiofencarb, fenobucarb, formetanate,furathiocarb, isoprocarb, methiocarb, methymyl, metolcarb, oxamyl,primicarb, propoxur, thiodicarb, thiofanox, triazamate, trimethacarb,XMC, xylylcarb, acephate, azamethiphos, azinphos-ethyl, azinphos-methyl,cadusafos, chlorethoxyfox, trichlorfon, vamidothion, chlordane,endosulfan, ethiprole, fipronil, acrinathrin, allethrin, bifenthrin,bioallethrin, bioalletherin X-cyclopentenyl, bioresmethrin,cyclorothrin, cyfluthrin, cyhalothrin, cypermethrin, cyphenothrin[(1R)-trans-isomers], deltamethrin, empenthrin [(EZ)-(1R)-isomers],esfenvalerate, etofenprox, fenpropathrin, fenvalerate, flucythrinate,flumethrin, halfenprox, kadathrin, phenothrin [(1R)-trans-isomer]prallethrin, pyrethrins (pyrethrum), resmethrin, silafluofen,tefluthrin, tetramethrin, tetramethrin [(1R)-isomers], tralomethrin,transfluthrin, alpha-cypermethrin, beta-cyfluthrin, beta-cypermethrin,d-cis-trans allethrin, d-trans allethrin, gamma-cyhalothrin,lamda-cyhalothrin, tau-fluvalinate, theta-cypermethrin,zeta-cypermethrin, methoxychlor, nicotine, sulfoxaflor, acetamiprid,clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid,thiamethoxan, tebuprimphos, beta-cyfluthrin, clothianidin, flonicamid,hydramethylnon, amitraz, flubendiamide, blorantraniliprole, lambdacyhalothrin, spinosad, gamma cyhalothrin, Beauveria bassiana, capsicumoleoresin extract, garlic oil, carbaryl, chlorpyrifos, sulfoxaflor,lambda cyhalothrin, Chlorfenvinphos, Chlormephos, Chlorpyrifos,Chlorpyrifos-methyl, Coumaphos, Cyanophos, Demeton-S-methyl, Diazinon,Dichlorvos/DDVP, Dicrotophos, Dimethoate, Dimethylvinphos, Disulfoton,EPN, Ethion, Ethoprophos, Famphur, Fenamiphos, Fenitrothion, Fenthion,Fosthiazate, Heptenophos, Imicyafos, Isofenphos, IsopropylO-(methoxyaminothio-phosphoryl) salicylate, Isoxathion, Malathion,Mecarbam, Methamidophos, Methidathion, Mevinphos, Monocrotophos, Naled,Omethoate, Oxydemeton-methyl, Parathion, Parathion-methyl, Phenthoate,Phorate, Phosalone, Phosmet, Phosphamidon, Phoxim, Pirimiphos-methyl,Profenofos, Propetamphos, Prothiofos, Pyraclofos, Pyridaphenthion,Quinalphosfluacrypyrim, tebufenozide, chlorantraniliprole, Bacillusthuringiensis subs. Kurstaki, terbufos, mineral oil, fenpropathrin,metaldehyde, deltamethrin, diazinon, dimethoate, diflubenzuron,pyriproxyfen, reosemary oil, peppermint oil, geraniol, azadirachtin,piperonyl butoxide, cyantraniliprole, alpha cypermethrin, tefluthrin,pymetrozine, malathion, Bacillus thuringiensis subsp. israelensis,dicofol, bromopropylate, benzoximate, azadirachtin, flonicamid, soybeanoil, Chromobacterium subtsugae strain PRAA4-1, zeta cypermethrin,phosmet, methoxyfenozide, paraffinic oil, spirotetramat, methomyl,Metarhizium anisopliae strain F52, ethoprop, tetradifon, propargite,fenbutatin oxide, azocyclotin, cyhexatin, diafenthiuron, Bacillussphaericus, etoxazole, flupyradifurone, azadirachtin, Beauveriabassiana, cyflumetofen, azadirachtin, chinomethionat, acephate, Isariafumosorosea Apopka strain 97, sodium tetraborohydrate decahydrate,emamectin benzoate, cryolite, spinetoram, Chenopodium ambrosioidesextract, novaluron, dinotefuran, carbaryl, acequinocyl, flupyradifurone,iron phosphate, kaolin, buprofezin, cyromazine, chromafenozide,halofenozide, methoxyfenozide, tebufenozide, bistrifluron,chlorfluazuron, diflubenzuron, flucycloxuron, flufenoxuron,hexaflumuron, lufenuron, nocaluron, noviflumuron, teflubenzuron,triflumuron, bensultap, cartap hydrochloride, thiocyclam,thiosultap-sodium, DNOC, chlorfenapyr, sulfuramid, phorate, tolfenpyrad,sulfoxaflor, neem oil, Bacillus thuringiensis subsp. tenebrionis strainSA-10, cyromazine, heat-killed Burkholderia spp., cyantraniliprole,cyenopyrafen, cyflumetofen, sodium cyanide, potassium cyanide, calciumcyanide, aluminum phosphide, calcium phosphide, phosphine, zincphosphide, spriodiclofen, spiromesifen, spirotetramat, metaflumizone,flubendiamide, pyflubumide, oxamyl, Bacillus thuringiensis subsp.aizawai, etoxazole, and esfenvalerate

TABLE 9 Exemplary insecticides associated with various modes of action,which can be combined with microbes of the disclosure PhysiologicalExemplary function(s) Mode of Action Compound class insecticidesaffected acetylcholinesterase carbamates Alanycarb, Aldicarb, Nerve and(AChE) inhibitors Bendiocarb, Benfuracarb, muscle Butocarboxim,Butoxycarboxim, Carbaryl, Carbofuran, Carbosulfan, Ethiofencarb,Fenobucarb, Formetanate, Furathiocarb, Isoprocarb, Methiocarb, Methomyl,Metolcarb, Oxamyl, Pirimicarb, Propoxur, Thiodicarb, Thiofanox,Triazamate, Trimethacarb, XMC, Xylylcarb acetylcholinesteraseorganophosphates Acephate, Azamethiphos, Nerve and (AChE) inhibitorsAzinphos-ethyl, Azinphos- muscle methyl, Cadusafos, Chlorethoxyfos,Chlorfenvinphos, Chlormephos, Chlorpyrifos, Chlorpyrifos-methyl,Coumaphos, Cyanophos, Demeton-S-methyl, Diazinon, Dichlorvos/DDVP,Dicrotophos, Dimethoate, Dimethylvinphos, Disulfoton, EPN, Ethion,Ethoprophos, Famphur, Fenamiphos, Fenitrothion, Fenthion, Fosthiazate,Heptenophos, Imicyafos, Isofenphos, Isopropyl O- (methoxyaminothio-phosphoryl) salicylate, Isoxathion, Malathion, Mecarbam, Methamidophos,Methidathion, Mevinphos, Monocrotophos, Naled, Omethoate, Oxydemeton-methyl, Parathion, Parathion- methyl, Phenthoate, Phorate, Phosalone,Phosmet, Phosphamidon, Phoxim, Pirimiphos-methyl, Profenofos,Propetamphos, Prothiofos, Pyraclofos, Pyridaphenthion, Quinalphos,Sulfotep, Tebupirimfos, Temephos, Terbufos, Tetrachlorvinphos,Thiometon, Triazophos, Trichlorfon, Vamidothion GABA-gated cyclodieneChlordane, Endosulfan Nerve and chloride channel organochlorines muscleblockers GABA-gated phenylpyrazoles Ethiprole, Fipronil Nerve andchloride channel (Fiproles) muscle blockers sodium channel pyrethroids,Acrinathrin, Allethrin, Nerve and modulators pyrethrins Bifenthrin,Bioallethrin, muscle Bioallethrin S-cyclopentenyl, Bioresmethrin,Cycloprothrin, Cyfluthrin, Cyhalothrin, Cypermethrin, Cyphenothrin[(1R)-trans-isomers], Deltamethrin, Empenthrin [(EZ)-(1R)-isomers],Esfenvalerate, Etofenprox, Fenpropathrin, Fenvalerate, Flucythrinate,Flumethrin, Halfenprox, Kadathrin, Phenothrin [(1R)-trans- isomer],Prallethrin, Pyrethrins (pyrethrum), Resmethrin, Silafluofen,Tefluthrin, Tetramethrin, Tetramethrin [(1R)-isomers], Tralomethrin,Transfluthrin, alpha- Cypermethrin, beta-Cyfluthrin, beta-Cypermethrin,d-cis-trans Allethrin, d-trans Allethrin, gamma-Cyhalothrin, lambda-Cvhalothrin, tau-Fluvalinate, theta-Cypermethrin, zeta- Cypermethrinsodium channel DDT, DDT, methoxychlor Nerve and modulators methoxychlormuscle nicotinic neonicotinoids Acetamiprid, Clothianidin, Nerve andacetylcholine Dinotefuran, Imidacloprid, muscle receptor (nAChR)Nitenpyram, Thiacloprid, competitive Thiamethoxam modulators nicotinicnicotine nicotine Nerve and acetylcholine muscle receptor (nAChR)competitive modulators nicotinic sulfoximines sulfoxaflor Nerve andacetylcholine muscle receptor (nAChR) competitive modulators nicotinicbutenolides Flupyradifurone Nerve and acetylcholine muscle receptor(nAChR) competitive modulators nicotinic spinosyns Spinetoram, SpinosadNerve and acetylcholine muscle receptor (nAChR) allosteric modulatorsGlutamate-gated avermectins, Abamectin, Emamectin Nerve and chloridechannel milbemycins benzoate, Lepimectin, muscle (GluCl) allostericMilbemectin modulators juvenile hormone juvenile hormone Hydroprene,Kinoprene, Growth mimics analogues Methoprene juvenile hormoneFenoxycarb Fenoxycarb Growth mimics juvenile hormone PyriproxyfenPyriproxyfen Growth mimics miscellaneous non- alkyl halides Methylbromide and other Unknown or specific (multi-site) alkyl halidesnon-specific inhibitors miscellaneous non- Chloropicrin ChloropicrinUnknown or specific (multi-site) non-specific inhibitors miscellaneousnon- fluorides Cryolite, sulfuryl fluoride Unknown or specific(multi-site) non-specific inhibitors miscellaneous non- borates Borax,Boric acid, Disodium Unknown or specific (multi-site) octaborate, Sodiumborate, non-specific inhibitors Sodium metaborate miscellaneous non-tartar emetic tartar emetic Unknown or specific (multi-site)non-specific inhibitors miscellaneous non- methyl Dazomet, Metam Unknownor specific (multi-site) isothiocyanate non-specific inhibitorsgenerators modulators of Pyridine Pymetrozine, Pyrifluquinazon Nerve andchordotonal organs azomethine muscle derivatives mite growthClofentezine, Clofentezine, Diflovidazin, Growth inhibitorsDiflovidazin, Hexythiazox Hexythiazox mite growth Etoxazole EtoxazoleGrowth inhibitors microbial Bacillus Bt var. aizawai, Bt var. Midgutdisruptors of insect thuringiensis and israelensis, Bt var. kurstaki, Btmidgut membranes the insecticidal var. tenebrionensis proteins theyproduce microbial Bacillus Bacillus sphaericus Midgut disruptors ofinsect sphaericus midgut membranes inhibitors of DiafenthiuronDiafenthiuron Respiration mitochondrial ATP synthase inhibitors oforganotin Azocyclotin, Cyhexatin, Respiration mitochondrial ATPmiticides Fenbutatin oxide synthase inhibitors of Propargite PropargiteRespiration mitochondrial ATP synthase inhibitors of TetradifonTetradifon Respiration mitochondrial ATP synthase uncouplers ofChlorfenapyr, Chlorfenapyr, DNOC, Respiration oxidative DNOC, Sulfuramidphosphorylation via Sulfuramid disruption of the proton gradientNicotinic nereistoxin Bensultap, Cartap Nerve and acetylcholineanalogues hydrochloride, Thiocyclam, muscle receptor (nAChR)Thiosultap-sodium channel blockers inhibitors of chitin benzoylureasBistrifluron, Chlorfluazuron, Growth biosynthesis, type 0 Diflubenzuron,Flucycloxuron, Flufenoxuron, Hexaflumuron, Lufenuron, Novaluron,Noviflumuron, Teflubenzuron, Triflumuron inhibitors of chitin BuprofezinBuprofezin Growth biosynthesis, type 1 moulting disruptor, CyromazineCyromazine Growth Dipteran ecdysone receptor diacylhydrazinesChromafenozide, Growth agonists Halofenozide, Methoxyfenozide,Tebufenozide octopamine Amitraz Amitraz Nerve and receptor agonistsmuscle mitochondrial Hydramethylnon Hydramethylnon Respiration complexIII electron transport inhibitors mitochondrial Acequinocyl AcequinocylRespiration complex III electron transport inhibitors mitochondrialFluacrypyrim Fluacrypyrim Respiration complex III electron transportinhibitors mitochondrial Bifenazate Bifenazate Respiration complex IIIelectron transport inhibitors mitochondrial Meti acaricides Fenazaquin,Fenpyroximate, Respiration complex I electron and insecticidesPyridaben, Pyrimidifen, transport inhibitors Tebufenpyrad, Tolfenpyradmitochondrial Rotenone Rotenone Respiration complex I electron transportinhibitors voltage-dependent oxadiazines Indoxacarb Nerve and sodiumchannel muscle blockers voltage-dependent semicarbazones MetaflumizoneNerve and sodium channel muscle blockers inhibitors of acetyl tetronicand Spirodiclofen, Spiromesifen, Growth CoA carboxylase tetramic acidSpirotetramat derivatives mitochondrial phosphides Aluminium phosphide,Respiration complex IV Calcium phosphide, electron transport Phosphine,Zinc phosphide inhibitors mitochondrial cyanides Calcium cyanide,Respiration complex IV Potassium cyanide, electron transport Sodiumcyanide inhibitors mitochondrial beta-ketonitrile Cyenopyrafen,Cyflumetofen Respiration complex II electron derivatives transportinhibitors mitochondrial carboxanilides Pyflubumide Respiration complexII electron transport inhibitors ryanodine receptor diamidesChlorantraniliprole, Nerve and modulators Cyantraniliprole, muscleFlubendiamide Chordotonal organ Flonicamid Flonicamid Nerve andmodulators - muscle undefined target site compounds of AzadirachtinAzadirachtin Unknown unknown or uncertain mode of action compounds ofBenzoximate Benzoximate Unknown unknown or uncertain mode of actioncompounds of Bromopropylate Bromopropylate Unknown unknown or uncertainmode of action compounds of Chinomethionat Chinomethionat Unknownunknown or uncertain mode of action compounds of Dicofol Dicofol Unknownunknown or uncertain mode of action compounds of lime sulfur lime sulfurUnknown unknown or uncertain mode of action compounds of PyridalylPyridalyl Unknown unknown or uncertain mode of action compounds ofsulfur sulfur Unknown unknown or uncertain mode of action

TABLE 10 Exemplary list of pesticides, which can be combined withmicrobes of the disclosure Category Compounds INSECTICIDES arsenicalinsecticides calcium arsenate copper acetoarsenite copper arsenate leadarsenate potassium arsenite sodium arsenite botanical insecticidesallicin anabasine azadirachtin carvacrol d-limonene matrine nicotinenornicotine oxymatrine pyrethrins cinerins cinerin I cinerin II jasmolinI jasmolin II pyrethrin I pyrethrin II quassia rhodojaponin-III rotenoneryania sabadilla sanguinarine triptolide carbamate insecticidesbendiocarb carbaryl benzofuranyl methylcarbamate insecticidesbenfuracarb carbofuran carbosulfan decarbofuran furathiocarbdimethylcarbamate insecticides dimetan dimetilan hyquincarb isolanpirimicarb pyramat pyrolan oxime carbamate insecticides alanycarbaldicarb aldoxycarb butocarboxim butoxycarboxim methomyl nitrilacarboxamyl tazimcarb thiocarboxime thiodicarb thiofanox phenylmethylcarbamate insecticides allyxycarb aminocarb bufencarb butacarbcarbanolate cloethocarb CPMC dicresyl dimethacarb dioxacarb EMPCethiofencarb fenethacarb fenobucarb isoprocarb methiocarb metolcarbmexacarbate promacyl promecarb propoxur trimethacarb XMC xylylcarbdiamide insecticides broflanilide chlorantraniliprole cyantraniliprolecyclaniliprole cyhalodiamide flubendiamide tetraniliprole dinitrophenolinsecticides dinex dinoprop dinosam DNOC fluorine insecticides bariumhexafluorosilicate cryolite flursulamid sodium fluoride sodiumhexafluorosilicate sulfluramid formamidine insecticides amitrazchlordimeform formetanate formparanate medimeform semiamitraz fumigantinsecticides acrylonitrile carbon disulfide carbon tetrachloridecarbonyl sulfide chloroform chloropicrin cyanogen para-dichlorobenzene1,2-dichloropropane dithioether ethyl formate ethylene dibromideethylene dichloride ethylene oxide hydrogen cyanide methyl bromidemethyl iodide methylchloroform methylene chloride naphthalene phosphinesodium tetrathiocarbonate sulfuryl fluoride tetrachloroethane inorganicinsecticides borax boric acid calcium polysulfide copper oleatediatomaceous earth mercurous chloride potassium thiocyanate silica gelsodium thiocyanate insect growth regulators chitin synthesis inhibitorsbuprofezin cyromazine benzoylphenylurea chitin synthesis bistrifluroninhibitors chlorbenzuron chlorfluazuron dichlorbenzuron diflubenzuronflucycloxuron flufenoxuron hexaflumuron lufenuron novaluron noviflumuronpenfluron teflubenzuron triflumuron juvenile hormone mimics dayoutongepofenonane fenoxycarb hydroprene kinoprene methoprene pyriproxyfentriprene juvenile hormones juvenile hormone I juvenile hormone IIjuvenile hormone III moulting hormone agonists chromafenozide furantebufenozide halofenozide methoxyfenozide tebufenozide yishijingmoulting hormones α-ecdysone ecdysterone moulting inhibitors diofenolanprecocenes precocene I precocene II precocene III unclassified insectgrowth regulators dicyclanil macrocyclic lactone insecticides avermectininsecticides abamectin doramectin emamectin eprinomectin ivermectinselamectin milbemycin insecticides lepimectin milbemectin milbemycinoxime moxidectin spinosyn insecticides spinetoram spinosad neonicotinoidinsecticides nitroguanidine neonicotinoid insecticides clothianidindinotefuran imidacloprid imidaclothiz thiamethoxam nitromethyleneneonicotinoid insecticides nitenpyram nithiazine pyridylmethylamineneonicotinoid acetamiprid insecticides imidacloprid nitenpyrampaichongding thiacloprid nereistoxin analogue insecticides bensultapcartap polythialan thiocyclam thiosultap organochlorine insecticidesbromo-DDT camphechlor DDT pp′-DDT ethyl-DDD HCH gamma-HCH lindanemethoxychlor pentachlorophenol TDE cyclodiene insecticides aldrinbromocyclen chlorbicyclen chlordane chlordecone dieldrin dilorendosulfan alpha-endosulfan endrin HEOD heptachlor HHDN isobenzanisodrin kelevan mirex organophosphorus insecticides organophosphateinsecticides bromfenvinfos calvinphos chlorfenvinphos crotoxyphosdichlorvos dicrotophos dimethylvinphos fospirate heptenophosmethocrotophos mevinphos monocrotophos naled naftalofos phosphamidonpropaphos TEPP tetrachlorvinphos organothiophosphate insecticidesdioxabenzofos fosmethilan phenthoate aliphatic organothiophosphateinsecticides acethion acetophos amiton cadusafos chlorethoxyfoschlormephos demephion demephion-O demephion-S demeton demeton-Odemeton-S demeton-methyl demeton-O-methyl demeton-S-methyldemeton-S-methylsulphon disulfoton ethion ethoprophos IPSP isothioatemalathion methacrifos methylacetophos oxydemeton-methyl oxydeprofosoxydisulfoton phorate sulfotep terbufos thiometon aliphatic amideorganothiophosphate amidithion insecticides cyanthoate dimethoateethoate-methyl formothion mecarbam omethoate prothoate sophamidevamidothion oxime organothiophosphate insecticides chlorphoxim phoximphoxim-methyl heterocyclic organothiophosphate azamethiphos insecticidescolophonate coumaphos coumithoate dioxathion endothion menazonmorphothion phosalone pyraclofos pyrazothion pyridaphenthion quinothionbenzothiopyran organothiophosphate dithicrofos insecticides thicrofosbenzotriazine organothiophosphate azinphos-ethyl insecticidesazinphos-methyl isoindole organothiophosphate insecticides dialifosphosmet isoxazole organothiophosphate insecticides isoxathion zolaprofospyrazolopyrimidine organothiophosphate chlorprazophos insecticidespyrazophos pyridine organothiophosphate insecticides chlorpyrifoschlorpyrifos-methyl pyrimidine organothiophosphate butathiofosinsecticides diazinon etrimfos lirimfos pirimioxyphos pirimiphos-ethylpirimiphos-methyl primidophos pyrimitate tebupirimfos quinoxalineorganothiophosphate quinalphos insecticides quinalphos-methylthiadiazole organothiophosphate athidathion insecticides lythidathionmethidathion prothidathion triazole organothiophosphate insecticidesisazofos triazophos phenyl organothinphosphate insecticides azothoatebromophos bromophos-ethyl carbophenothion chlorthiophos cyanophoscythioate dicapthon dichlofenthion etaphos famphur fenchlorphosfenitrothion fensulfothion fenthion fenthion-ethyl heterophos jodfenphosmesulfenfos parathion parathion-methyl phenkapton phosnichlor profenofosprothiofos sulprofos temephos trichlormetaphos-3 trifenofosxiaochongliulin phosphonate insecticides butonate trichlorfonphosphonothioate insecticides mecarphon phenyl ethylphosphonothioateinsecticides fonofos trichloronat phenyl phenylphosphonothioatecyanofenphos insecticides EPN leptophos phosphoramidate insecticidescrufomate fenamiphos fosthietan mephosfolan phosfolan phosfolan-methylpirimetaphos phosphoramidothioate insecticides acephate chloraminephosphorus isocarbophos isofenphos isofenphos-methyl methamidophosphosglycin propetamphos phosphorodiamide insecticides dimefox mazidoxmipafox schradan oxadiazine insecticides indoxacarb oxadiazoloneinsecticides metoxadiazone phthalimide insecticides dialifos phosmettetramethrin physical insecticides maltodextrin desiccant insecticidesboric acid diatomaceous earth silica gel pyrazole insecticideschlorantraniliprole cyantraniliprole cyclaniliprole dimetilan isolantebufenpyrad tetraniliprole tolfenpyrad phenylpyrazole insecticidesacetoprole ethiprole fipronil flufiprole pyraclofos pyrafluprolepyriprole pyrolan vaniliprole pyrethroid insecticides pyrethroid esterinsecticides acrinathrin allethrin bioallethrin esdépalléthrine barthrinbifenthrin kappa-bifenthrin bioethanomethrin brofenvaleratebrofluthrinate bromethrin butethrin chlorempenthrin cyclethrincycloprothrin cyfluthrin beta-cyfluthrin cyhalothrin gamma-cyhalothrinlambda-cyhalothrin cypermethrin alpha-cypermethrin beta-cypermethrintheta-cypermethrin zeta-cypermethrin cyphenothrin deltamethrindimefluthrin dimethrin empenthrin d-fanshiluquebingjuzhichloroprallethrin fenfluthrin fenpirithrin fenpropathrin fenvalerateesfenvalerate flucythrinate fluvalinate tau-fluvalinate furamethrinfurethrin heptafluthrin imiprothrin japothrins kadethrin methothrinmetofluthrin epsilon-metofluthrin momfluorothrin epsilon-momfluorothrinpentmethrin permethrin biopermethrin transpermethrin phenothrinprallethrin profluthrin proparthrin pyresmethrin renofluthrinmeperfluthrin resmethrin bioresmethrin cismethrin tefluthrinkappa-tefluthrin terallethrin tetramethrin tetramethylfluthrintralocythrin tralomethrin transfluthrin valerate pyrethroid etherinsecticides etofenprox flufenprox halfenprox protrifenbute silafluofenpyrethroid oxime insecticides sulfoxime thiofluoximate pyrimidinamineinsecticides flufenerim pyrimidifen pyrrole insecticides chlorfenapyrquaternary ammonium insecticides sanguinarine sulfoximine insecticidessulfoxaflor tetramic acid insecticides spirotetramat tetronic acidinsecticides spiromesifen thiazole insecticides clothianidinimidaclothiz thiamethoxam thiapronil thiazolidine insecticides tazimcarbthiacloprid thiourea insecticides diafenthiuron urea insecticidesflucofuron sulcofuron zwitterionic insecticides dicloromezotiaztriflumezopyrim unclassified insecticides afidopyropen afoxolanerallosamidin closantel copper naphthenate crotamiton EXD fenazaflorfenoxacrim flometoquin flonicamid fluhexafon flupyradifurone fluralanerfluxametamide hydramethylnon isoprothiolane jiahuangchongzong malonobenmetaflumizone nifluridide plifenate pyridaben pyridalyl pyrifluquinazonrafoxanide thuringiensin triarathene triazamate ACARICIDES botanicalacaricides carvacrol sanguinarine bridged diphenyl acaricides azobenzenebenzoximate benzyl benzoate bromopropylate chlorbenside chlorfenetholchlorfenson chlorfensulphide chlorobenzilate chloropropylatecyflumetofen DDT dicofol diphenyl sulfone dofenapyn fenson fentrifanilfluorbenside genit hexachlorophene phenproxide proclonol tetradifontetrasul carbamate acaricides benomyl carbanolate carbaryl carbofuranmethiocarb metolcarb promacyl propoxur oxime carbamate acaricidesaldicarb butocarboxim oxamyl thiocarboxime thiofanox carbazateacaricides bifenazate dinitrophenol acaricides binapacryl dinexdinobuton dinocap dinocap-4 dinocap-6 dinocton dinopenton dinosulfondinoterbon DNOC formamidine acaricides amitraz chlordimeformchloromebuform formetanate formparanate medimeform semiamitrazmacrocyclic lactone acaricides tetranactin avermectin acaricidesabamectin doramectin eprinomectin ivermectin selamectin milbemycinacaricides milbemectin milbemycin oxime moxidectin mite growthregulators clofentezine cyromazine diflovidazin dofenapyn fluazuronflubenzimine flucycloxuron flufenoxuron hexythiazox organochlorineacaricides bromocyclen camphechlor DDT dienochlor endosulfan lindaneorganophosphorus acaricides organophosphate acaricides chlorfenvinphoscrotoxyphos dichlorvos heptenophos mevinphos monocrotophos naled TEPPtetrachlorvinphos organothiophosphate acaricides amidithion amitonazinphos-ethyl azinphos-methyl azothoate benoxafos bromophosbromophos-ethyl carbophenothion chlorpyrifos chlorthiophos coumaphoscyanthoate demeton demeton-O demeton-S demeton-methyl demeton-O-methyldemeton-S-methyl demeton-S-methylsulphon dialifos diazinon dimethoatedioxathion disulfoton endothion ethion ethoate-methyl formothionmalathion mecarbam methacrifos omethoate oxydeprofos oxydisulfotonparathion phenkapton phorate phosalone phosmet phostin phoximpirimiphos-methyl prothidathion prothoate pyrimitate quinalphosqumtiofos sophamide sulfotep thiometon triazophos trifenofos vamidothionphosphonate acaricides trichlorfon phosphoramidothioate acaricidesisocarbophos methamidophos propetamphos phosphorodiamide acaricidesdimefox mipafox schradan organotin acaricides azocyclotin cyhexatinfenbutatin oxide phostin phenylsulfamide acaricides dichlofluanidphthalimide acaricides dialifos phosmet pyrazole acaricides cyenopyrafenfenpyroximate pyflubumide tebufenpyrad phenylpyrazole acaricidesacetoprole fipronil vaniliprole pyrethroid acaricides pyrethroid esteracaricides acrinathrin bifenthrin brofluthrinate cyhalothrincypermethrin alpha-cypermethrin fenpropathrin fenvalerate flucythrinateflumethrin fluvalinate tau-fluvalinate permethrin pyrethroid etheracaricides halfenprox pyrimidinamine acaricides pyrimidifen pyrroleacaricides chlorfenapyr quaternary ammonium acaricides sanguinarinequinoxaline acaricides chinomethionat thioquinox strobilurin acaricidesmethoxyacrylate strobilurin acaricides bifujunzhi fluacrypyrimflufenoxystrobin pyriminostrobin sulfite ester acaricides aramitepropargite tetronic acid acaricides spirodiclofen tetrazine acaricidesclofentezine diflovidazin thiazolidine acaricides flubenziminehexythiazox thiocarbamate acaricides fenothiocarb thiourea acaricideschloromethiuron diafenthiuron unclassified acaricides acequinocylafoxolaner amidoflumet arsenous oxide clenpirin closantel crotamitoncycloprate cymiazole disulfiram etoxazole fenazaflor fenazaquinfluenetil fluralaner mesulfen MNAF nifluridide nikkomycins pyridabensulfiram sulfluramid sulfur thuringiensin triarathene CHEMOSTERILANTSapholate bisazir busulfan diflubenzuron dimatif hemel hempa metepamethiotepa methyl apholate morzid penfluron tepa thiohempa thiotepatretamine uredepa INSECT REPELLENTS acrep butopyronoxyl camphord-camphor carboxide dibutyl phthalate diethyltoluamide dimethyl carbatedimethyl phthalate dibutyl succinate ethohexadiol hexamide icaridinmethoquin-butyl methylneodecanamide 2-(octylthio)ethanol oxamatequwenzhi quyingding rebemide zengxiaoan NEMATICIDES avermectinnematicides abamectin botanical nematicides carvacrol carbamatenematicides benomyl carbofuran carbosulfan cloethocarb oxime carbamatenematicides alanycarb aldicarb aldoxycarb oxamyl tirpate fumigantnematicides carbon disulfide cyanogen 1,2-dichloropropane1,3-dichloropropene dithioether methyl bromide methyl iodide sodiumtetrathiocarbonate organophosphorus nematicides organophosphatenematicides diamidafos fenamiphos fosthietan phosphamidonorganothiophosphate nematicides cadusafos chlorpyrifos dichlofenthiondimethoate ethoprophos fensulfothion fosthiazate heterophos isamidofosisazofos phorate phosphocarb terbufos thionazin triazophosphosphonothioate nematicides imicyafos mecarphon unclassifiednematicides acetoprole benclothiaz chloropicrin dazomet DBCP DCIPfluazaindolizine fluensulfone furfural metam methyl isothiocyanatetioxazafen xylenols

Insecticides also include synergists or activators that are not inthemselves considered toxic or insecticidal, but are materials used withinsecticides to synergize or enhance the activity of the insecticides.Synergists or activators include piperonyl butoxide.

Biorational Pesticides

Insecticides can be biorational, or can also be known as biopesticidesor biological pesticides. Biorational refers to any substance of naturalorigin (or man-made substances resembling those of natural origin) thathas a detrimental or lethal effect on specific target pest(s), e.g.,insects, weeds, plant diseases (including nematodes), and vertebratepests, possess a unique mode of action, are non-toxic to man, domesticplants and animals, and have little or no adverse effects on wildlifeand the environment.

Biorational insecticides (or biopesticides or biological pesticides) canbe grouped as: (1) biochemicals (hormones, enzymes, pheromones andnatural agents, such as insect and plant growth regulators), (2)microbial (viruses, bacteria, fungi, protozoa, and nematodes), or (3)Plant-Incorporated protectants (PIPs)—primarily transgenic plants, e.g.,Bt corn.

Biopesticides, or biological pesticides, can broadly include agentsmanufactured from living microorganisms or a natural product and soldfor the control of plant pests. Biopesticides can be: microorganisms,biochemicals, and semiochemicals. Biopesticides can also includepeptides, proteins and nucleic acids such as double-stranded DNA,single-stranded DNA, double-stranded RNA, single-stranded RNA andhairpin DNA or RNA.

Bacteria, fungi, oomycetes, viruses and protozoa are all used for thebiological control of insect pests. The most widely used microbialbiopesticide is the insect pathogenic bacteria Bacillus thuringiensis(Bt), which produces a protein crystal (the Bt δ-endotoxin) duringbacterial spore formation that is capable of causing lysis of gut cellswhen consumed by susceptible insects. Microbial Bt biopesticides consistof bacterial spores and δ-endotoxin crystals mass-produced infermentation tanks and formulated as a sprayable product. Bt does notharm vertebrates and is safe to people, beneficial organisms and theenvironment. Thus, Bt sprays are a growing tactic for pest management onfruit and vegetable crops where their high level of selectivity andsafety are considered desirable, and where resistance to syntheticchemical insecticides is a problem. Bt sprays have also been used oncommodity crops such as maize, soybean and cotton, but with the adventof genetic modification of plants, farmers are increasingly growing Bttransgenic crop varieties.

Other microbial insecticides include products based on entomopathogenicbaculoviruses. Baculoviruses that are pathogenic to arthropods belong tothe virus family and possess large circular, covalently closed, anddouble-stranded DNA genomes that are packaged into nucleocapsids. Morethan 700 baculoviruses have been identified from insects of the ordersLepidoptera, Hymenoptera, and Diptera. Baculoviruses are usually highlyspecific to their host insects and thus, are safe to the environment,humans, other plants, and beneficial organisms. Over 50 baculovirusproducts have been used to control different insect pests worldwide. Inthe US and Europe, the Cydia pomonella granulovirus (CpGV) is used as aninundative biopesticide against codling moth on apples. WashingtonState, as the biggest apple producer in the US, uses CpGV on 13% of theapple crop. In Brazil, the nucleopolyhedrovirus of the soybeancaterpillar Anticarsia gemmatalis was used on up to 4 million ha(approximately 35%) of the soybean crop in the mid-1990s. Viruses suchas Gemstar® (Certis USA) are available to control larvae of Heliothisand Helicoverpa species.

At least 170 different biopesticide products based on entomopathogenicfungi have been developed for use against at least five insect andacarine orders in glasshouse crops, fruit and field vegetables as wellas commodity crops. The majority of products are based on theascomycetes Beauveria bassiana or Metarhizium anisopliae. M anisopliaehas also been developed for the control of locust and grasshopper pestsin Africa and Australia and is recommended by the Food and AgricultureOrganization of the United Nations (FAO) for locust management.

A number of microbial pesticides registered in the United States arelisted in Table 16 of Kabaluk et al. 2010 (Kabaluk, J. T. et al. (ed.).2010. The Use and Regulation of Microbial Pesticides in RepresentativeJurisdictions Worldwide. IOBC Global. 99pp.) and microbial pesticidesregistered in selected countries are listed in Annex 4 ofHoeschle-Zeledon et al. 2013 (Hoeschle-Zeledon, I., P. Neuenschwanderand L. Kumar. (2013). Regulatory Challenges for biological control.SP-IPM Secretariat, International Institute of Tropical Agriculture(IITA), Ibadan, Nigeria. 43 pp.), each of which is incorporated hereinin its entirety.

Plants produce a wide variety of secondary metabolites that deterherbivores from feeding on them. Some of these can be used asbiopesticides. They include, for example, pyrethrins, which arefast-acting insecticidal compounds produced by Chrysanthemumcinerariaefolium. They have low mammalian toxicity but degrade rapidlyafter application. This short persistence prompted the development ofsynthetic pyrethrins (pyrethroids). The most widely used botanicalcompound is neem oil, an insecticidal chemical extracted from seeds ofAzadirachta indica. Two highly active pesticides are available based onsecondary metabolites synthesized by soil actinomycetes, but they havebeen evaluated by regulatory authorities as if they were syntheticchemical pesticides. Spinosad is a mixture of two macrolide compoundsfrom Saccharopolyspora spinosa. It has a very low mammalian toxicity andresidues degrade rapidly in the field. Farmers and growers used itwidely following its introduction in 1997 but resistance has alreadydeveloped in some important pests such as western flower thrips.Abamectin is a macrocyclic lactone compound produced by Streptomycesavermitilis. It is active against a range of pest species but resistancehas developed to it also, for example, in tetranychid mites.

Peptides and proteins from a number of organisms have been found topossess pesticidal properties. Perhaps most prominent are peptides fromspider venom (King, G. F. and Hardy, M. C. (2013) Spider-venom peptides:structure, pharmacology, and potential for control of insect pests.Annu. Rev. Entomol. 58: 475-496). A unique arrangement of disulfidebonds in spider venom peptides render them extremely resistant toproteases. As a result, these peptides are highly stable in the insectgut and hemolymph and many of them are orally active. The peptidestarget a wide range of receptors and ion channels in the insect nervoussystem. Other examples of insecticidal peptides include: sea anemonevenom that act on voltage-gated Na+ channels (Bosmans, F. and Tytgat, J.(2007) Sea anemone venom as a source of insecticidal peptides acting onvoltage-gated Na+ channels. Toxicon. 49(4): 550-560); the PA1b (PeaAlbumin 1, subunit b) peptide from Legume seeds with lethal activity onseveral insect pests, such as mosquitoes, some aphids and cereal weevils(Eyraud, V. et al. (2013) Expression and Biological Activity of theCystine Knot Bioinsecticide PA1b (Pea Albumin 1 Subunit b). PLoS ONE8(12): e81619); and an internal 10 kDa peptide generated by enzymatichydrolysis of Canavalia ensiformis (jack bean) urease within susceptibleinsects (Martinelli, A. H. S., et al. (2014) Structure—function studieson jaburetox, a recombinant insecticidal peptide derived from jack bean(Canavalia ensiformis) urease. Biochimica et Biophysica Acta 1840:935-944). Examples of commercially available peptide insecticidesinclude Spear™—T for the treatment of thrips in vegetables andornamentals in greenhouses, Spear™—P to control the Colorado PotatoBeetle, and Spear™—C to protect crops from lepidopteran pests (VestaronCorporation, Kalamazoo, Mich.). A novel insecticidal protein fromBacillus bombysepticus, called parasporal crystal toxin (PC), shows oralpathogenic activity and lethality towards silkworms and Cry1Ac-resistantHelicoverpa armigera strains (Lin, P. et al. (2015) PC, a novel oralinsecticidal toxin from Bacillus bombysepticus involved in hostlethality via APN and BtR-175. Sci. Rep. 5: 11101).

A semiochemical is a chemical signal produced by one organism thatcauses a behavioral change in an individual of the same or a differentspecies. The most widely used semiochemicals for crop protection areinsect sex pheromones, some of which can now be synthesized and are usedfor monitoring or pest control by mass trapping, lure-and-kill systemsand mating disruption. Worldwide, mating disruption is used on over660,000 ha and has been particularly useful in orchard crops.

As used herein, “transgenic insecticidal trait” refers to a traitexhibited by a plant that has been genetically engineered to express anucleic acid or polypeptide that is detrimental to one or more pests. Inone embodiment, the plants of the present disclosure are resistant toattach and/or infestation from any one or more of the pests of thepresent disclosure. In one embodiment, the trait comprises theexpression of vegetative insecticidal proteins (VIPs) from Bacillusthuringiensis, lectins and proteinase inhibitors from plants,terpenoids, cholesterol oxidases from Streptomyces spp., insectchitinases and fungal chitinolytic enzymes, bacterial insecticidalproteins and early recognition resistance genes. In another embodiment,the trait comprises the expression of a Bacillus thuringiensis proteinthat is toxic to a pest. In one embodiment, the Bt protein is a Cryprotein (crystal protein). Bt crops include Bt corn, Bt cotton and Btsoy. Bt toxins can be from the Cry family (see, for example, Crickmoreet al., 1998, Microbiol. Mol. Biol. Rev. 62: 807-812), which areparticularly effective against Lepidoptera, Coleoptera and Diptera.

Bt Cry and Cyt toxins belong to a class of bacterial toxins known aspore-forming toxins (PFT) that are secreted as water-soluble proteinsundergoing conformational changes in order to insert into, or totranslocate across, cell membranes of their host. There are two maingroups of PFT: (i) the α-helical toxins, in which α-helix regions formthe trans-membrane pore, and (ii) the β-barrel toxins, that insert intothe membrane by forming a β-barrel composed of βsheet hairpins from eachmonomer. See, Parker M W, Feil S C, “Pore-forming protein toxins: fromstructure to function,” Prog. Biophys. Mol. Biol. 2005 May;88(1):91-142. The first class of PFT includes toxins such as thecolicins, exotoxin A, diphtheria toxin and also the Cry three-domaintoxins. On the other hand, aerolysin, α-hemolysin, anthrax protectiveantigen, cholesterol-dependent toxins as the perfringolysin O and theCyt toxins belong to the β-barrel toxins. Id. In general, PFTproducing-bacteria secrete their toxins and these toxins interact withspecific receptors located on the host cell surface. In most cases, PFTare activated by host proteases after receptor binding inducing theformation of an oligomeric structure that is insertion competent.Finally, membrane insertion is triggered, in most cases, by a decreasein pH that induces a molten globule state of the protein. Id.

The development of transgenic crops that produce Bt Cry proteins hasallowed the substitution of chemical insecticides by environmentallyfriendly alternatives. In transgenic plants the Cry toxin is producedcontinuously, protecting the toxin from degradation and making itreachable to chewing and boring insects. Cry protein production inplants has been improved by engineering cry genes with a plant biasedcodon usage, by removal of putative splicing signal sequences anddeletion of the carboxy-terminal region of the protoxin. See, Schuler TH, et al., “Insect-resistant transgenic plants,” Trends Biotechnol.1998; 16:168-175. The use of insect resistant crops has diminishedconsiderably the use of chemical pesticides in areas where thesetransgenic crops are planted. See, Qaim M, Zilberman D, “Yield effectsof genetically modified crops in developing countries,” Science. 2003Feb. 7; 299(5608):900-2.

Known Cry proteins include: δ-endotoxins including but not limited to:the Cry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11,Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21,Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry 28, Cry 29, Cry 30, Cry31,Cry32, Cry33, Cry34, Cry35, Cry36, Cry37, Cry38, Cry39, Cry40, Cry41,Cry42, Cry43, Cry44, Cry45, Cry 46, Cry47, Cry49, Cry 51, Cry52, Cry 53,Cry 54, Cry55, Cry56, Cry57, Cry58, Cry59. Cry60, Cry61, Cry62, Cry63,Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70 and Cry71 classes ofδ-endotoxin genes and the B. thuringiensis cytolytic cyt1 and cyt2genes.

Members of these classes of B. thuringiensis insecticidal proteinsinclude, but are not limited to: Cry1Aa1 (Accession #AAA22353); Cry1Aa2(Accession #Accession #AAA22552); Cry1Aa3 (Accession #BAA00257); Cry1Aa4(Accession #CAA31886); Cry1Aa5 (Accession #BAA04468); Cry1Aa6 (Accession#AAA86265); Cry1Aa7 (Accession #AAD46139); Cry1Aa8 (Accession #126149);Cry1Aa9 (Accession #BAA77213); Cry1Aa10 (Accession #AAD55382); Cry1Aa1 1(Accession #CAA70856); Cry1Aa12 (Accession #AAP80146); Cry1Aa13(Accession #AAM44305); Cry1Aa14 (Accession #AAP40639); Cry1Aa15(Accession #AAY66993); Cry1Aa16 (Accession #HQ439776); Cry1Aa17(Accession #HQ439788); Cry1Aa18 (Accession #HQ439790); Cry1Aa19(Accession #HQ685121); Cry1Aa20 (Accession #JF340156); Cry1Aa21(Accession #JN651496); Cry1Aa22 (Accession #KC158223); Cry1Ab1(Accession #AAA22330); Cry1Ab2 (Accession #AAA22613); Cry1Ab3 (Accession#AAA22561); Cry1Ab4 (Accession #BAA00071); Cry1Ab5 (Accession#CAA28405); Cry1Ab6 (Accession #AAA22420); Cry1Ab7 (Accession#CAA31620); Cry1Ab8 (Accession #AAA22551); Cry1Ab9 (Accession#CAA38701); Cry1Ab10 (Accession #A29125); Cry1Ab11 (Accession #112419);Cry1Ab12 (Accession #AAC64003); Cry1Ab13 (Accession #AAN76494); Cry1Ab14(Accession #AAG16877); Cry1Ab15 (Accession #AA013302); Cry1Ab16(Accession #AAK55546); Cry1Ab17 (Accession #AAT46415); Cry1Ab18(Accession #AAQ88259); Cry1Ab19 (Accession #AAW31761); Cry1Ab20(Accession #ABB72460); Cry1Ab21 (Accession #ABS18384); Cry1Ab22(Accession #ABW87320); Cry1Ab23 (Accession #HQ439777); Cry1Ab24(Accession #HQ439778); Cry1Ab25 (Accession #HQ685122); Cry1Ab26(Accession #HQ847729); Cry1Ab27 (Accession #JN135249); Cry1Ab28(Accession #JN135250); Cry1Ab29 (Accession #JN135251); Cry1Ab30(Accession #JN135252); Cry1Ab31 (Accession #JN135253); Cry1Ab32(Accession #JN135254); Cry1Ab33 (Accession #AAS93798); Cry1Ab34(Accession #KC156668); Cry1Ab-like (Accession #AAK14336); Cry1Ab-like(Accession #AAK14337); Cry1Ab-like (Accession #AAK14338); Cry1Ab-like(Accession #ABG88858); Cry1Ac1 (Accession #AAA22331); Cry1Ac2 (Accession#AAA22338); Cry1Ac3 (Accession #CAA38098); Cry1Ac4 (Accession#AAA73077); Cry1Ac5 (Accession #AAA22339); Cry1Ac6 (Accession#AAA86266); Cry1Ac7 (Accession #AAB46989); Cry1Ac8 (Accession#AAC44841); Cry1Ac9 (Accession #AAB49768); Cry1Ac10 (Accession#CAA05505); Cry1Ac11 (Accession #CAA10270); Cry1Ac12 (Accession#112418); Cry1Ac13 (Accession #AAD38701); Cry1Ac14 (Accession#AAQ06607); Cry1Ac15 (Accession #AAN07788); Cry1Ac16 (Accession#AAU87037); Cry1Ac17 (Accession #AAX18704); Cry1Ac18 (Accession#AAY88347); Cry1Ac19 (Accession #ABD37053); Cry1Ac20 (Accession#ABB89046); Cry1Ac21 (Accession #AAY66992); Cry1Ac22 (Accession#ABZ01836); Cry1Ac23 (Accession #CAQ30431); Cry1Ac24 (Accession#ABL01535); Cry1Ac25 (Accession #FJ513324); Cry1Ac26 (Accession#FJ617446); Cry1Ac27 (Accession #FJ617447); Cry1Ac28 (Accession#ACM90319); Cry1Ac29 (Accession #DQ438941); Cry1Ac30 (Accession#GQ227507); Cry1Ac31 (Accession #GU446674); Cry1Ac32 (Accession#HM061081); Cry1Ac33 (Accession #GQ866913); Cry1Ac34 (Accession#HQ230364); Cry1Ac35 (Accession #JF340157); Cry1Ac36 (Accession#JN387137); Cry1Ac37 (Accession #JQ317685); Cry1Ad1 (Accession#AAA22340); Cry1Ad2 (Accession #CAA01880); Cry1Ae1 (Accession#AAA22410); Cry1Af1 (Accession #AAB82749); Cry1Ag1 (Accession#AAD46137); Cry1Ah1 (Accession #AAQ14326); Cry1Ah2 (Accession#ABB76664); Cry1Ah3 (Accession #HQ439779); Cry1Ai1 (Accession#AA039719); Cry1Ai2 (Accession #HQ439780); Cry1A-like (Accession#AAK14339); Cry1Ba1 (Accession #CAA29898); Cry1Ba2 (Accession#CAA65003); Cry1Ba3 (Accession #AAK63251); Cry1Ba4 (Accession#AAK51084); Cry1Ba5 (Accession #AB020894); Cry1Ba6 (Accession#ABL60921); Cry1Ba7 (Accession #HQ439781); Cry1Bb1 (Accession#AAA22344); Cry1Bb2 (Accession #HQ439782); Cry1Bc1 (Accession#CAA86568); Cry1Bd1 (Accession #AAD10292); Cry1Bd2 (Accession#AAM93496); Cry1Be1 (Accession #AAC32850); Cry1Be2 (Accession#AAQ52387); Cry1Be3 (Accession #ACV96720); Cry1Be4 (Accession#HM070026); Cry1Bf1 (Accession #CAC50778); Cry1Bf2 (Accession#AAQ52380); Cry1Bg1 (Accession #AA039720); Cry1Bh1 (Accession#HQ589331); Cry1Bi1 (Accession #KC156700); Cry1Ca1 (Accession#CAA30396); Cry1Ca2 (Accession #CAA31951); Cry1Ca3 (Accession#AAA22343); Cry1Ca4 (Accession #CAA01886); Cry1Ca5 (Accession#CAA65457); Cry1Ca6 [1] (Accession #AAF37224); Cry1Ca7 (Accession#AAG50438); Cry1Ca8 (Accession #AAM00264); Cry1Ca9 (Accession#AAL79362); Cry1Ca10 (Accession #AAN16462); Cry1Ca11 (Accession#AAX53094); Cry1Ca12 (Accession #HM070027); Cry1Ca13 (Accession#HQ412621); Cry1Ca14 (Accession #JN651493); Cry1Cb1 (Accession #M97880);Cry1Cb2 (Accession #AAG35409); Cry1Cb3 (Accession #ACD50894);Cry1Cb-like (Accession #AAX63901); Cry1Da1 (Accession #CAA38099);Cry1Da2 (Accession #176415); Cry1Da3 (Accession #HQ439784); Cry1 Db1(Accession #CAA80234); Cry1 Db2 (Accession #AAK48937); Cry1 Dc1(Accession #ABK35074); Cry1Ea1 (Accession #CAA37933); Cry1Ea2 (Accession#CAA39609); Cry1Ea3 (Accession #AAA22345); Cry1Ea4 (Accession#AAD04732); Cry1Ea5 (Accession #A15535); Cry1Ea6 (Accession #AAL50330);Cry1Ea7 (Accession #AAW72936); Cry1Ea8 (Accession #ABX11258); Cry1Ea9(Accession #HQ439785); Cry1Ea10 (Accession #ADR00398); Cry1Ea11(Accession #JQ652456); Cry1Eb1 (Accession #AAA22346); Cry1Fa1 (Accession#AAA22348); Cry1Fa2 (Accession #AAA22347); Cry1Fa3 (Accession#HM070028); Cry1Fa4 (Accession #HM439638); Cry1 Fb1 (Accession#CAA80235); Cry1Fb2 (Accession #BAA25298); Cry1Fb3 (Accession#AAF21767); Cry1Fb4 (Accession #AAC10641); Cry1Fb5 (Accession#AA013295); Cry1Fb6 (Accession #ACD50892); Cry1Fb7 (Accession#ACD50893); Cry1Ga1 (Accession #CAA80233); Cry1 Ga2 (Accession#CAA70506); Cry1Gb1 (Accession #AAD10291); Cry1Gb2 (Accession#AA013756); Cry1Gc1 (Accession #AAQ52381); Cry1Ha1 (Accession#CAA80236); Cry1Hb1 (Accession #AAA79694); Cry1Hb2 (Accession#HQ439786); Cry1H-like (Accession #AAF01213); Cry1Ia1 (Accession#CAA44633); Cry1Ia2 (Accession #AAA22354); Cry1Ia3 (Accession#AAC36999); Cry1Ia4 (Accession #AAB00958); Cry1Ia5 (Accession#CAA70124); Cry1Ia6 (Accession #AAC26910); Cry1Ia7 (Accession#AAM73516); Cry1Ia8 (Accession #AAK66742); Cry1Ia9 (Accession#AAQ08616); Cry1Ia10 (Accession #AAP86782); Cry1Ia11 (Accession#CAC85964); Cry1Ia12 (Accession #AAV53390); Cry1Ia13 (Accession#ABF83202); Cry1Ia14 (Accession #ACG63871); Cry1Ia15 (Accession#FJ617445); Cry1Ia16 (Accession #FJ617448); Cry1Ia17 (Accession#GU989199); Cry1Ia18 (Accession #ADK23801); Cry1Ia19 (Accession#HQ439787); Cry1Ia20 (Accession #JQ228426); Cry1Ia21 (Accession#JQ228424); Cry1Ia22 (Accession #JQ228427); Cry1Ia23 (Accession#JQ228428); Cry1Ia24 (Accession #JQ228429); Cry1Ia25 (Accession#JQ228430); Cry1Ia26 (Accession #JQ228431); Cry1Ia27 (Accession#JQ228432); Cry1Ia28 (Accession #JQ228433); Cry1Ia29 (Accession#JQ228434); Cry1Ia30 (Accession #JQ317686); Cry1Ia31 (Accession#JX944038); Cry1Ia32 (Accession #JX944039); Cry1Ia33 (Accession#JX944040); Cry1Ib1 (Accession #AAA82114); Cry1Ib2 (Accession#ABW88019); Cry1Ib3 (Accession #ACD75515); Cry1Ib4 (Accession#HM051227); Cry1Ib5 (Accession #HM070028); Cry1Ib6 (Accession#ADK38579); Cry1Ib7 (Accession #JN571740); Cry1Ib8 (Accession#JN675714); Cry1Ib9 (Accession #JN675715); Cry1Ib10 (Accession#JN675716); Cry1Ib11 (Accession #JQ228423); Cry1Ic1 (Accession#AAC62933); Cry1Ic2 (Accession #AAE71691); Cry1Id1 (Accession#AAD44366); Cry1Id2 (Accession #JQ228422); Cry1Ie1 (Accession#AAG43526); Cry1Ie2 (Accession #HM439636); Cry1Ie3 (Accession#KC156647); Cry1Ie4 (Accession #KC156681); Cry1If1 (Accession#AAQ52382); Cry1Ig1 (Accession #KC156701); Cry1I-like (Accession#AAC31094); Cry1I-like (Accession #ABG88859); Cry1Ja1 (Accession#AAA22341); Cry1Ja2 (Accession #HM070030); Cry1Ja3 (Accession#JQ228425); Cry1Jb1 (Accession #AAA98959); Cry1Jc1 (Accession#AAC31092); Cry1Jc2 (Accession #AAQ52372); Cry1Jd1 (Accession#CAC50779); Cry1Ka1 (Accession #AAB00376); Cry1Ka2 (Accession#HQ439783); Cry1La1 (Accession #AAS60191); Cry1La2 (Accession#HM070031); Cry1Ma1 (Accession #FJ884067); Cry1Ma2 (Accession#KC156659); Cry1Na1 (Accession #KC156648); Cry1Nb1 (Accession#KC156678); Cry1-like (Accession #AAC31091); Cry2Aa1 (Accession#AAA22335); Cry2Aa2 (Accession #AAA83516); Cry2Aa3 (Accession #D86064);Cry2Aa4 (Accession #AAC04867); Cry2Aa5 (Accession #CAA10671); Cry2Aa6(Accession #CAA10672); Cry2Aa7 (Accession #CAA10670); Cry2Aa8 (Accession#AA013734); Cry2Aa9 (Accession #AA013750); Cry2Aa1 O (Accession#AAQ04263); Cry2Aa1 1 (Accession #AAQ52384); Cry2Aa12 (Accession#AB183671); Cry2Aa13 (Accession #ABL01536); Cry2Aa14 (Accession#ACF04939); Cry2Aa15 (Accession #JN426947); Cry2Ab1 (Accession#AAA22342); Cry2Ab2 (Accession #CAA39075); Cry2Ab3 (Accession#AAG36762); Cry2Ab4 (Accession #AA013296); Cry2Ab5 (Accession#AAQ04609); Cry2Ab6 (Accession #AAP59457); Cry2Ab7 (Accession#AAZ66347); Cry2Ab8 (Accession #ABC95996); Cry2Ab9 (Accession#ABC74968); Cry2Ab10 (Accession #EF157306); Cry2Ab11 (Accession#CAM84575); Cry2Ab12 (Accession #ABM21764); Cry2Ab13 (Accession#ACG76120); Cry2Ab14 (Accession #ACG76121); Cry2Ab15 (Accession#HM037126); Cry2Ab16 (Accession #GQ866914); Cry2Ab1 7 (Accession#HQ439789); Cry2Ab18 (Accession #JN135255); Cry2Ab19 (Accession#JN135256); Cry2Ab20 (Accession #JN135257); Cry2Ab21 (Accession#JN135258); Cry2Ab22 (Accession #JN135259); Cry2Ab23 (Accession#JN135260); Cry2Ab24 (Accession #JN135261); Cry2Ab25 (Accession#JN415485); Cry2Ab26 (Accession #JN426946); Cry2Ab27 (Accession#JN415764); Cry2Ab28 (Accession #JN651494); Cry2Ac1 (Accession#CAA40536); Cry2Ac2 (Accession #AAG35410); Cry2Ac3 (Accession#AAQ52385); Cry2Ac4 (Accession #ABC95997); Cry2Ac5 (Accession#ABC74969); Cry2Ac6 (Accession #ABC74793); Cry2Ac7 (Accession#CAL18690); Cry2Ac8 (Accession #CAM09325); Cry2Ac9 (Accession#CAM09326); Cry2Ac10 (Accession #ABN15104); Cry2Ac11 (Accession#CAM83895); Cry2Ac12 (Accession #CAM83896); Cry2Ad1 (Accession#AAF09583); Cry2Ad2 (Accession #ABC86927); Cry2Ad3 (Accession#CAK29504); Cry2Ad4 (Accession #CAM32331); Cry2Ad5 (Accession#CA078739); Cry2Ae1 (Accession #AAQ52362); Cry2Af1 (Accession#AB030519); Cry2Af2 (Accession #GQ866915); Cry2Ag1 (Accession#ACH91610); Cry2Ah1 (Accession #EU939453); Cry2Ah2 (Accession#ACL80665); Cry2Ah3 (Accession #GU073380); Cry2Ah4 (Accession#KC156702); Cry2Ai1 (Accession #FJ788388); Cry2Aj (Accession #); Cry2Ak1(Accession #KC156660); Cry2Ba1 (Accession #KC156658); Cry3Aa1 (Accession#AAA22336); Cry3Aa2 (Accession #AAA22541); Cry3Aa3 (Accession#CAA68482); Cry3Aa4 (Accession #AAA22542); Cry3Aa5 (Accession#AAA50255); Cry3Aa6 (Accession #AAC43266); Cry3Aa7 (Accession#CAB41411); Cry3Aa8 (Accession #AAS79487); Cry3Aa9 (Accession#AAW05659); Cry3Aa10 (Accession #AAU29411); Cry3Aa11 (Accession#AAW82872); Cry3Aa12 (Accession #ABY49136); Cry3Ba1 (Accession#CAA34983); Cry3Ba2 (Accession #CAA00645); Cry3Ba3 (Accession#JQ397327); Cry3Bb1 (Accession #AAA22334); Cry3Bb2 (Accession#AAA74198); Cry3Bb3 (Accession #115475); Cry3Ca1 (Accession #CAA42469);Cry4Aa1 (Accession #CAA68485); Cry4Aa2 (Accession #BAAOO1 79); Cry4Aa3(Accession #CAD30148); Cry4Aa4 (Accession #AFB18317); Cry4A-like(Accession #AAY96321); Cry4Ba1 (Accession #CAA30312); Cry4Ba2 (Accession#CAA30114); Cry4Ba3 (Accession #AAA22337); Cry4Ba4 (Accession #BAAOO178); Cry4Ba5 (Accession #CAD30095); Cry4Ba-like (Accession #ABC47686);Cry4Ca1 (Accession #EU646202); Cry4Cb1 (Accession #FJ403208); Cry4Cb2(Accession #FJ597622); Cry4Cc1 (Accession #FJ403207); Cry5Aa1 (Accession#AAA67694); Cry5Ab1 (Accession #AAA67693); Cry5Ac1 (Accession #134543);Cry5Ad1 (Accession #ABQ82087); Cry5Ba1 (Accession #AAA68598); Cry5Ba2(Accession #ABW88931); Cry5Ba3 (Accession #AFJ04417); Cry5Ca1 (Accession#HM461869); Cry5Ca2 (Accession #ZP_04123426); Cry5Da1 (Accession#HM461870); Cry5Da2 (Accession #ZP_04123980); Cry5Ea1 (Accession#HM485580); Cry5Ea2 (Accession #ZP_04124038); Cry6Aa1 (Accession#AAA22357); Cry6Aa2 (Accession #AAM46849); Cry6Aa3 (Accession#ABH03377); Cry6Ba1 (Accession #AAA22358); Cry7 Aa1 (Accession#AAA22351); Cry7Ab1 (Accession #AAA21120); Cry7Ab2 (Accession#AAA21121); Cry7Ab3 (Accession #ABX24522); Cry7 Ab4 (Accession#EU380678); Cry7 Ab5 (Accession #ABX79555); Cry7 Ab6 (Accession#ACI44005); Cry7 Ab7 (Accession #ADB89216); Cry7 Ab8 (Accession#GU145299); Cry7Ab9 (Accession #ADD92572); Cry7Ba1 (Accession#ABB70817); Cry7Bb1 (Accession #KC156653); Cry7Ca1 (Accession#ABR67863); Cry7Cb1 (Accession #KC156698); Cry7Da1 (Accession#ACQ99547); Cry7Da2 (Accession #HM572236); Cry7Da3 (Accession#KC156679); Cry7Ea1 (Accession #HM035086); Cry7Ea2 (Accession#HM132124); Cry7Ea3 (Accession #EEM19403); Cry7Fa1 (Accession#HM035088); Cry7Fa2 (Accession #EEM19090); Cry7Fb1 (Accession#HM572235); Cry7Fb2 (Accession #KC156682); Cry7Ga1 (Accession#HM572237); Cry7Ga2 (Accession #KC156669); Cry7Gb1 (Accession#KC156650); Cry7Gc1 (Accession #KC156654); Cry7Gd1 (Accession#KC156697); Cry7Ha1 (Accession #KC156651); Cry7Ia1 (Accession#KC156665); Cry7Ja1 (Accession #KC156671); Cry7Ka1 (Accession#KC156680); Cry7Kb1 (Accession #BAM99306); Cry7La1 (Accession#BAM99307); Cry8Aa1 (Accession #AAA21117); Cry8Ab1 (Accession#EU044830); Cry8Ac1 (Accession #KC156662); Cry8Ad1 (Accession#KC156684); Cry8Ba1 (Accession #AAA21118); Cry8Bb1 (Accession#CAD57542); Cry8Bc1 (Accession #CAD57543); Cry8Ca1 (Accession#AAA21119); Cry8Ca2 (Accession #AAR98783); Cry8Ca3 (Accession#EU625349); Cry8Ca4 (Accession #ADB54826); Cry8Da1 (Accession#BAC07226); Cry8Da2 (Accession #BD133574); Cry8Da3 (Accession#BD133575); Cry8Db1 (Accession #BAF93483); Cry8Ea1 (Accession#AAQ73470); Cry8Ea2 (Accession #EU047597); Cry8Ea3 (Accession#KC855216); Cry8Fa1 (Accession #AAT48690); Cry8Fa2 (Accession#HQ174208); Cry8Fa3 (Accession #AFH78109); Cry8Ga1 (Accession#AAT46073); Cry8Ga2 (Accession #ABC42043); Cry8Ga3 (Accession#FJ198072); Cry8Ha1 (Accession #AAW81032); Cry8Ia1 (Accession#EU381044); Cry8Ia2 (Accession #GU073381); Cry8Ia3 (Accession#HM044664); Cry8Ia4 (Accession #KC156674); Cry8Ib1 (Accession#GU325772); Cry8Ib2 (Accession #KC156677); Cry8Ja1 (Accession#EU625348); Cry8Ka1 (Accession #FJ422558); Cry8Ka2 (Accession#ACN87262); Cry8Kb1 (Accession #HM123758); Cry8Kb2 (Accession#KC156675); Cry8La1 (Accession #GU325771); Cry8Ma1 (Accession#HM044665); Cry8Ma2 (Accession #EEM86551); Cry8Ma3 (Accession#HM210574); Cry8Na1 (Accession #HM640939); Cry8Pa1 (Accession#HQ388415); Cry8Qa1 (Accession #HQ441166); Cry8Qa2 (Accession#KC152468); Cry8Ra1 (Accession #AFP87548); Cry8Sa1 (Accession#JQ740599); Cry8Ta1 (Accession #KC156673); Cry8-like (Accession#FJ770571); Cry8-like (Accession #ABS53003); Cry9Aa1 (Accession#CAA41122); Cry9Aa2 (Accession #CAA41425); Cry9Aa3 (Accession#GQ249293); Cry9Aa4 (Accession #GQ249294); Cry9Aa5 (Accession #JX174110); Cry9Aa like (Accession #AAQ52376); Cry9Ba1 (Accession#CAA52927); Cry9Ba2 (Accession #GU299522); Cry9Bb1 (Accession#AAV28716); Cry9Ca1 (Accession #CAA85764); Cry9Ca2 (Accession#AAQ52375); Cry9Da1 (Accession #BAA1 9948); Cry9Da2 (Accession#AAB97923); Cry9Da3 (Accession #GQ249293); Cry9Da4 (Accession#GQ249297); Cry9Db1 (Accession #AAX78439); Cry9Dc1 (Accession #KCl56683); Cry9Ea1 (Accession #BAA34908); Cry9Ea2 (Accession #AA012908);Cry9Ea3 (Accession #ABM21765); Cry9Ea4 (Accession #ACE88267); Cry9Ea5(Accession #ACF04743); Cry9Ea6 (Accession #ACG63872); Cry9Ea7 (Accession#FJ380927); Cry9Ea8 (Accession #GQ249292); Cry9Ea9 (Accession#JN651495); Cry9Eb1 (Accession #CAC50780); Cry9Eb2 (Accession#GQ249298); Cry9Eb3 (Accession #KC156646); Cry9Ec1 (Accession#AAC63366); Cry9Ed1 (Accession #AAX78440); Cry9Ee1 (Accession#GQ249296); Cry9Ee2 (Accession #KC156664); Cry9Fa1 (Accession#KC156692); Cry9Ga1 (Accession #KC156699); Cry9-like (Accession#AAC63366); Cry1OAa1 (Accession #AAA22614); Cry10Aa2 (Accession#E00614); Cry10Aa3 (Accession #CAD30098); Cry10Aa4 (Accession#AFB18318); Cry1OA-like (Accession #DQ167578); Cry1 1Aa1 (Accession#AAA22352); Cry1 1Aa2 (Accession #AAA22611); Cry1 1Aa3 (Accession#CAD30081); Cry1 1Aa4 (Accession #AFB18319); Cry11Aa-like (Accession#DQ166531); Cry11Ba1 (Accession #CAA60504); Cry11Bb1 (Accession#AAC97162); Cry1 1Bb2 (Accession #HM068615); Cry12Aa1 (Accession#AAA22355); Cry13Aa1 (Accession #AAA22356); Cry14Aa1 (Accession#AAA21516); Cry14Ab1 (Accession #KC156652); Cry15Aa1 (Accession#AAA22333); Cry16Aa1 (Accession #CAA63860); Cry17Aa1 (Accession#CAA67841); Cry18Aa1 (Accession #CAA67506); Cry18Ba1 (Accession#AAF89667); Cry18Ca1 (Accession #AAF89668); Cry19Aa1 (Accession#CAA68875); Cry19Ba1 (Accession #BAA32397); Cry19Ca1 (Accession#AFM37572); Cry20Aa1 (Accession #AAB93476); Cry20Ba1 (Accession#ACS93601); Cry20Ba2 (Accession #KC156694); Cry20-like (Accession#GQ144333); Cry21Aa1 (Accession #132932); Cry21Aa2 (Accession #166477);Cry21Ba1 (Accession #BAC06484); Cry21Ca1 (Accession #JF521577); Cry21Ca2(Accession #KC156687); Cry21Da1 (Accession #JF521578); Cry22Aa1(Accession #134547); Cry22Aa2 (Accession #CAD43579); Cry22Aa3 (Accession#ACD93211); Cry22Ab1 (Accession #AAK50456); Cry22Ab2 (Accession#CAD43577); Cry22Ba1 (Accession #CAD43578); Cry22Bb1 (Accession#KC156672); Cry23Aa1 (Accession #AAF76375); Cry24Aa1 (Accession#AAC61891); Cry24Ba1 (Accession #BAD32657); Cry24Ca1 (Accession#CAJ43600); Cry25Aa1 (Accession #AAC61892); Cry26Aa1 (Accession#AAD25075); Cry27Aa1 (Accession #BAA82796); Cry28Aa1 (Accession#AAD24189); Cry28Aa2 (Accession #AAG00235); Cry29Aa1 (Accession#CAC80985); Cry30Aa1 (Accession #CAC80986); Cry30Ba1 (Accession#BAD00052); Cry30Ca1 (Accession #BAD67157); Cry30Ca2 (Accession#ACU24781); Cry30Da1 (Accession #EF095955); Cry30Db1 (Accession#BAE80088); Cry30Ea1 (Accession #ACC95445); Cry30Ea2 (Accession#FJ499389); Cry30Fa1 (Accession #ACI22625); Cry30Ga1 (Accession#ACG60020); Cry30Ga2 (Accession #HQ638217); Cry31Aa1 (Accession #BAB11757); Cry31Aa2 (Accession #AAL87458); Cry31Aa3 (Accession #BAE79808);Cry31Aa4 (Accession #BAF32571); Cry31Aa5 (Accession #BAF32572); Cry31Aa6(Accession #BA144026); Cry31Ab1 (Accession #BAE79809); Cry31Ab2(Accession #BAF32570); Cry31Ac1 (Accession #BAF34368); Cry31Ac2(Accession #AB731600); Cry31Ad1 (Accession #BA144022); Cry32Aa1(Accession #AAG36711); Cry32Aa2 (Accession #GU063849); Cry32Ab1(Accession #GU063850); Cry32Ba1 (Accession #BAB78601); Cry32Ca1(Accession #BAB78602); Cry32Cb1 (Accession #KC156708); Cry32Da1(Accession #BAB78603); Cry32Ea1 (Accession #GU324274); Cry32Ea2(Accession #KC156686); Cry32Eb1 (Accession #KC156663); Cry32Fa1(Accession #KC156656); Cry32Ga1 (Accession #KC156657); Cry32Ha1(Accession #KC156661); Cry32Hb1 (Accession #KC156666); Cry32Ia1(Accession #KCl 56667); Cry32Ja1 (Accession #KCl 56685); Cry32Ka1(Accession #KCl 56688); Cry32La1 (Accession #KC156689); Cry32Ma1(Accession #KC156690); Cry32Mb1 (Accession #KC156704); Cry32Na1(Accession #KC156691); Cry32Oa1 (Accession #KC156703); Cry32Pa1(Accession #KC156705); Cry32Qa1 (Accession #KC156706); Cry32Ra1(Accession #KC156707); Cry32Sa1 (Accession #KC156709); Cry32Ta1(Accession #KC156710); Cry32Ua1 (Accession #KC156655); Cry33Aa1(Accession #AAL26871); Cry34Aa1 (Accession #AAG50341); Cry34Aa2(Accession #AAK64560); Cry34Aa3 (Accession #AAT29032); Cry34Aa4(Accession #AAT29030); Cry34Ab1 (Accession #AAG41671); Cry34Ac1(Accession #AAG50118); Cry34Ac2 (Accession #AAK64562); Cry34Ac3(Accession #AAT29029); Cry34Ba1 (Accession #AAK64565); Cry34Ba2(Accession #AAT29033); Cry34Ba3 (Accession #AAT29031); Cry35Aa1(Accession #AAG50342); Cry35Aa2 (Accession #AAK64561); Cry35Aa3(Accession #AAT29028); Cry35Aa4 (Accession #AAT29025); Cry35Ab1(Accession #AAG41672); Cry35Ab2 (Accession #AAK64563); Cry35Ab3(Accession #AY536891); Cry35Ac1 (Accession #AAG50117); Cry35Ba1(Accession #AAK64566); Cry35Ba2 (Accession #AAT29027); Cry35Ba3(Accession #AAT29026); Cry36Aa1 (Accession #AAK64558); Cry37 Aa1(Accession #AAF76376); Cry38Aa1 (Accession #AAK64559); Cry39Aa1(Accession #BAB72016); Cry40Aa1 (Accession #BAB72018); Cry40Ba1(Accession #BAC77648); Cry40Ca1 (Accession #EU381045); Cry40Da1(Accession #ACF15199); Cry41Aa1 (Accession #BAD35157); Cry41Ab1(Accession #BAD35163); Cry41Ba1 (Accession #HM461871); Cry41Ba2(Accession #ZP_04099652); Cry42Aa1 (Accession #BAD35166); Cry43Aa1(Accession #BAD15301); Cry43Aa2 (Accession #BAD95474); Cry43Ba1(Accession #BAD15303); Cry43Ca1 (Accession #KC156676); Cry43Cb1(Accession #KC156695); Cry43Cc1 (Accession #KC156696); Cry43-like(Accession #BAD15305); Cry44Aa (Accession #BAD08532); Cry45Aa (Accession#BAD22577); Cry46Aa (Accession #BAC79010); Cry46Aa2 (Accession#BAG68906); Cry46Ab (Accession #BAD35170); Cry47 Aa (Accession#AAY24695); Cry48Aa (Accession #CAJ18351); Cry48Aa2 (Accession#CAJ86545); Cry48Aa3 (Accession #CAJ86546); Cry48Ab (Accession#CAJ86548); Cry48Ab2 (Accession #CAJ86549); Cry49Aa (Accession#CAH56541); Cry49Aa2 (Accession #CAJ86541); Cry49Aa3 (Accession#CAJ86543); Cry49Aa4 (Accession #CAJ86544); Cry49Ab1 (Accession#CAJ86542); Cry50Aa1 (Accession #BAE86999); Cry50Ba1 (Accession#GU446675); Cry50Ba2 (Accession #GU446676); Cry51Aa1 (Accession#AB114444); Cry51Aa2 (Accession #GU570697); Cry52Aa1 (Accession#EF613489); Cry52Ba1 (Accession #FJ361760); Cry53Aa1 (Accession#EF633476); Cry53Ab1 (Accession #FJ361759); Cry54Aa1 (Accession#ACA52194); Cry54Aa2 (Accession #GQ140349); Cry54Ba1 (Accession#GU446677); Cry55Aa1 (Accession #ABW88932); Cry54Ab1 (Accession#JQ916908); Cry55Aa2 (Accession #AAE33526); Cry56Aa1 (Accession#ACU57499); Cry56Aa2 (Accession #GQ483512); Cry56Aa3 (Accession#JX025567); Cry57Aa1 (Accession #ANC87261); Cry58Aa1 (Accession#ANC87260); Cry59Ba1 (Accession #JN790647); Cry59Aa1 (Accession#ACR43758); Cry60Aa1 (Accession #ACU24782); Cry60Aa2 (Accession#EA057254); Cry60Aa3 (Accession #EEM99278); Cry60Ba1 (Accession#GU810818); Cry60Ba2 (Accession #EA057253); Cry60Ba3 (Accession#EEM99279); Cry61Aa1 (Accession #HM035087); Cry61Aa2 (Accession#HM132125); Cry61Aa3 (Accession #EEM19308); Cry62Aa1 (Accession#HM054509); Cry63Aa1 (Accession #BA144028); Cry64Aa1 (Accession#BAJ05397); Cry65Aa1 (Accession #HM461868); Cry65Aa2 (Accession#ZP_04123838); Cry66Aa1 (Accession #HM485581); Cry66Aa2 (Accession#ZP_04099945); Cry67Aa1 (Accession #HM485582); Cry67Aa2 (Accession#ZP_04148882); Cry68Aa1 (Accession #HQ113114); Cry69Aa1 (Accession#HQ401006); Cry69Aa2 (Accession #JQ821388); Cry69Ab1 (Accession#JN209957); Cry70Aa1 (Accession #JN646781); Cry70Ba1 (Accession#AD051070); Cry70Bb1 (Accession #EEL67276); Cry71Aa1 (Accession#JX025568); Cry72Aa1 (Accession #JX025569); Cyt1Aa (GenBank AccessionNumber X03182); Cyt1Ab (GenBank Accession Number X98793); Cyt1B (GenBankAccession Number U37196); Cyt2A (GenBank Accession Number Z14147); andCyt2B (GenBank Accession Number U52043).

Examples of δ-endotoxins also include but are not limited to Cry1Aproteins of U.S. Pat. Nos. 5,880,275, 7,858,849 8,530,411, 8,575,433,and 8,686,233; a DIG-3 or DIG-11 toxin (N-terminal deletion of a-helix 1and/or a-helix 2 variants of cry proteins such as Cry1A, Cry3A) of U.S.Pat. Nos. 8,304,604, 8,304,605 and 8,476,226; Cry1B of U.S. patentapplication Ser. No. 10/525,318; Cry1C of U.S. Pat. No. 6,033,874; Cry1Fof U.S. Pat. Nos. 5,188,960 and 6,218,188; Cry1A/F chimeras of U.S. Pat.Nos. 7,070,982; 6,962,705 and 6,713,063); a Cry2 protein such as Cry2Abprotein of U.S. Pat. No. 7,064,249); a Cry3A protein including but notlimited to an engineered hybrid insecticidal protein (eHIP) created byfusing unique combinations of variable regions and conserved blocks ofat least two different Cry proteins (US Patent Application PublicationNumber 2010/0017914); a Cry4 protein; a Cry5 protein; a Cry6 protein;Cry8 proteins of U.S. Pat. Nos. 7,329,736, 7,449,552, 7,803,943,7,476,781, 7,105,332, 7,378,499 and 7,462,760; a Cry9 protein such assuch as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E and Cry9Ffamilies, including but not limited to the Cry9D protein of U.S. Pat.No. 8,802,933 and the Cry9B protein of U.S. Pat. No. 8,802,934; a Cry15protein of Naimov, et al., (2008), “Applied and EnvironmentalMicrobiology,” 74:7145-7151; a Cry22, a Cry34Ab1 protein of U.S. Pat.Nos. 6,127,180, 6,624,145 and 6,340,593; a CryET33 and cryET34 proteinof U.S. Pat. Nos. 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107and 7,504,229; a CryET33 and CryET34 homologs of US Patent PublicationNumber 2006/0191034, 2012/0278954, and PCT Publication Number WO2012/139004; a Cry35Ab1 protein of U.S. Pat. Nos. 6,083,499, 6,548,291and 6,340,593; a Cry46 protein, a Cry 51 protein, a Cry binary toxin; aTIC901 or related toxin; TIC807 of US Patent Application PublicationNumber 2008/0295207; ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128of PCT US 2006/033867; TIC853 toxins of U.S. Pat. No. 8,513,494,AXMI-027, AXMI-036, and AXMI-038 of U.S. Pat. No. 8,236,757; AXMI-031,AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. No. 7,923,602; AXMI-018,AXMI-020 and AXMI-021 of WO 2006/083891; AXMI-010 of WO 2005/038032;AXMI-003 of WO 2005/021585; AXMI-008 of US Patent ApplicationPublication Number 2004/0250311; AXMI-006 of US Patent ApplicationPublication Number 2004/0216186; AXMI-007 of US Patent Applica-tionPublication Number 2004/0210965; AXMI-009 of US Patent ApplicationNumber 2004/0210964; AXMI-014 of US Patent Application PublicationNumber 2004/0197917; AXMI-004 of US Patent Application PublicationNumber 2004/0197916; AXMI-028 and AXMI-029 of WO 2006/119457; AXMI-007,AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO2004/074462; AXMI-150 of U.S. Pat. No. 8,084,416; AXMI-205 of US PatentApplication Publication Number 2011/0023184; AXMI-011, AXMI-012,AXMI-013, AXMI-015, AXMI-019, AXMI-044, AXMI-037, AXMI-043, AXMI-033,AXMI-034, AXMI-022, AXMI-023, AXMI-041, AXMI-063 and AXMI-064 of USPatent Application Publication Number 2011/0263488; AXMI-R1 and relatedproteins of US Patent Application Publication Number 2010/0197592;AXMI221Z, AXMI222z, AXMI223z, AXMI224z and AXMI225z of WO 2011/103248;AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230and AXMI231 of WO 2011/103247 and U.S. Pat. No. 8,759,619; AXMI-115,AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. No. 8,334,431;AXMI-001, AXMI-002, AXMI-030, AXMI-035 and AXMI-045 of US PatentApplication Publication Number 2010/0298211; AXMI-066 and AXMI-076 of USPatent Application Publication Number 2009/0144852; AXMI128, AXMI130,AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146,AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157,AXMI158, AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170,AXMI171, AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178,AXMI179, AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188,AXMI189 of U.S. Pat. No. 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082,AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101,AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111,AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121,AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129,AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 of USPatent Application Publication Number 2010/0005543, AXMI270 of US PatentApplication Publication US20140223598, AXMI279 of US Patent ApplicationPublication US20140223599, cry proteins such as Cry1A and Cry3A havingmodified proteolytic sites of U.S. Pat. No. 8,319,019; a Cry1Ac, Cry2Aaand Cry1 Ca toxin protein from Bacillus thuringiensis strain VBTS 2528of US Patent Application Publication Number 2011/0064710. Other Cryproteins are well known to one skilled in the art. See, N. Crickmore, etal., “Revision of the Nomenclature for the Bacillus thuringiensisPesticidal Crystal Proteins,” Microbiology and Molecular BiologyReviews,” (1998) Vol 62: 807-813; see also, N. Crickmore, et al.,“Bacillus thuringiensis toxin nomenclature” (2016), atwww.btnomenclature.info/.

The use of Cry proteins as transgenic plant traits is well known to oneskilled in the art and Cry-transgenic plants including but not limitedto plants expressing Cry1Ac, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F,Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1,Cry35Ab1, Vip3A, mCry3A, Cry9c and CBI-Bt have received regulatoryapproval. See, Sanahuja et al., “Bacillus thuringiensis: a century ofresearch, development and commercial applications,” (2011) Plant BiotechJournal, April 9(3):283-300 and the CERA (2010) GM Crop Database Centerfor Environmental Risk Assessment (CERA), ILSI Research Foundation,Washington D.C. at cera-gmc.org/index.php?action=gm_crop_database, whichcan be accessed on the world-wide web using the “www” prefix). More thanone pesticidal proteins well known to one skilled in the art can also beexpressed in plants such as Vip3Ab & Cry1Fa (US2012/0317682); Cry1BE &Cry1F (US2012/0311746); Cry1CA & Cry1AB (US2012/0311745); Cry1F & CryCa(US2012/0317681); Cry1DA& Cry1BE (US2012/0331590); Cry1DA & Cry1Fa(US2012/0331589); Cry1AB & Cry1BE (US2012/0324606); Cry1Fa & Cry2Aa andCry11 & Cry1E (US2012/0324605); Cry34Ab/35Ab and Cry6Aa (US20130167269);Cry34Ab/VCry35Ab & Cry3Aa (US20130167268); Cry1Ab & Cry1F(US20140182018); and Cry3A and Cry1Ab or Vip3Aa (US20130116170).Pesticidal proteins also include insecticidal lipases including lipidacyl hydrolases of U.S. Pat. No. 7,491,869, and cholesterol oxidasessuch as from Streptomyces (Purcell et al. (1993) Biochem Biophys ResCommun 15:1406-1413).

Pesticidal proteins also include VIP (vegetative insecticidal proteins)toxins. Entomopathogenic bacteria produce insecticidal proteins thataccumulate in inclusion bodies or parasporal crystals (such as theaforementioned Cry and Cyt proteins), as well as insecticidal proteinsthat are secreted into the culture medium. Among the latter are the Vipproteins, which are divided into four families according to their aminoacid identity. The Vip1 and Vip2 proteins act as binary toxins and aretoxic to some members of the Coleoptera and Hemiptera. The Vip1component is thought to bind to receptors in the membrane of the insectmidgut, and the Vip2 component enters the cell, where it displays itsADP-ribosyltransferase activity against actin, preventing microfilamentformation. Vip3 has no sequence similarity to Vip1 or Vip2 and is toxicto a wide variety of members of the Lepidoptera. Its mode of action hasbeen shown to resemble that of the Cry proteins in terms of proteolyticactivation, binding to the midgut epithelial membrane, and poreformation, although Vip3A proteins do not share binding sites with Cryproteins. The latter property makes them good candidates to be combinedwith Cry proteins in transgenic plants (Bacillus thuringiensis-treatedcrops [Bt crops]) to prevent or delay insect resistance and to broadenthe insecticidal spectrum. There are commercially grown varieties of Btcotton and Bt maize that express the Vip3Aa protein in combination withCry proteins. For the most recently reported Vip4 family, no targetinsects have been found yet. See, Chakroun et al., “Bacterial VegetativeInsecticidal Proteins (Vip) from Entomopathogenic Bacteria,” MicrobiolMol Biol Rev. 2016 Mar. 2; 80(2):329-50. VIPs can be found in U.S. Pat.Nos. 5,877,012, 6,107,279 6,137,033, 7,244,820, 7,615,686, and 8,237,020and the like. Other VIP proteins are well known to one skilled in theart (see, lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html, whichcan be accessed on the world-wide web using the “www” prefix).

Pesticidal proteins also include toxin complex (TC) proteins, obtainablefrom organisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see,U.S. Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins have “standalone” insecticidal activity and other TC proteins enhance the activityof the stand-alone toxins produced by the same given organism. Thetoxicity of a “stand-alone” TC protein (from Photorhabdus, Xenorhabdusor Paenibacillus, for example) can be enhanced by one or more TC protein“potentiators” derived from a source organism of a different genus.There are three main types of TC proteins. As referred to herein, ClassA proteins (“Protein A”) are stand-alone toxins. Class B proteins(“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity ofClass A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 andXptA2. Examples of Class B proteins are TcaC, TcdB, XptB1Xb and XptC1Wi. Examples of Class C proteins are TccC, XptC1Xb and XptB1 Wi.Pesticidal proteins also include spider, snake and scorpion venomproteins. Examples of spider venom peptides include, but are not limitedto lycotoxin-1 peptides and mutants thereof (U.S. Pat. No. 8,334,366).

Some currently registered PIPs are listed in Table 11. Transgenic plantshave also been engineered to express dsRNA directed against insect genes(Baum, J. A. et al. (2007) Control of coleopteran insect pests throughRNA interference. Nature Biotechnology 25: 1322-1326; Mao, Y. B. et al.(2007) Silencing a cotton bollworm P450 monooxygenase gene byplant-mediated RNAi impairs larval tolerance of gossypol. NatureBiotechnology 25: 1307-1313). RNA interference can be triggered in thepest by feeding of the pest on the transgenic plant. Pest feeding thuscauses injury or death to the pest.

TABLE 11 List of exemplary Plant-incorporated Protectants, which can becombined with microbes of the disclosure Plant-Incorporated Company andTrade Pesticide Registration Protectants (PIPs) Names Numbers PotatoPotato Cry3A Potato PC Code 006432 Naturemark 524-474 New Leaf MonsantoCry3A & PLRV Potato Monsanto 524-498 PC Codes 006432, 006469 New LeafPlus Corn Cry1Ab Corn Event 176 PC Code 006458 Mycogen Seeds/Dow 68467-1Agro 66736-1 Syngenta Seeds Cry1Ab Corn Event Bt11 EPA PC Code AgrisureCB (with 67979-1 006444 OECD Unique Identifier SYN- Yieldgard) 65268-1BTØ11-1, Attribute Insect Protected Sweet Corn Syngenta Seeds Cry1AbCorn Event MON 801 Monsanto 524-492 Cry1Ab corn Event MON 810 PC CodeMonsanto 524-489 006430 OECD Unique Identifier MON- ØØ81Ø-6 Cry1Ac CornPC Code 006463 Dekalb Genetics c/o 69575-2 Monsanto BT-XTRA Cry1F cornEvent TC1507 PC Code Mycogen Seeds/Dow 68467-2 006481 OECD UniqueIdentifier DAS- Agro 29964-3 Ø15Ø7-1 Pioneer Hi- Bred/Dupont moCry1Fcorn Event DAS-Ø6275-8 PC Mycogen Seeds/Dow 68467-4 Code 006491 OECDUnique Identifier Agro DAS-Ø6275-8 Cry9C Corn Aventis 264-669 StarLinkCry3Bb1 corn Event MON863 PC Code Monsanto 524-528 006484 YielGard RWOECD Unique Identifier MON-ØØ863-5 Cry3Bb1 corn Event MON 88017 PC CodeMonsanto 524-551 006498 YieldGrad VT OECD Unique Identifier MON-88Ø17-3Rootworm Cry34Ab1/Cry35Ab1 corn Event DAS- Mycogen Seeds/Dow 68467-5591227-7 Agro 29964-4 PC Code 006490 Pioneer Hi- OECD Unique IdentifierDAS-59122-7 Bred/Dupont Herculex Rootworm Cry34Ab1/Cry35Ab1 and Cry1Fcorn Pioneer Hi- 29964-17 Event 4114 Bred/Dupont PC Codes 006555, 006556mCry3A corn Event MIR 604 Syngenta Seeds 67979-5 PC Code 006509 OECDUnique Identifier Agrisure RW SYN-IR604-8 Cry1A.105 and Cry2Ab2 cornEvent MON Monsanto 524-575 89034 PC Codes 006515 and 006514 Genuity VTDouble Pro Vip3Aa20 corn Event MIR 162 Syngenta Seeds 67979-14 PC Code006599 OECD Unique Identifier Agrisure Viptera SYN-IR162-4 eCry3.1Abcorn in Event 5307 PC Code Syngenta 67979-22 016483 OECD UniqueIdentifier SYN- Æ53Æ7-1 Stacked Events and Seed Blend Corn MON863 ×MON810 with Cry3Bb1 + Monsanto YieldGard 524-545 Cry1Ab Plus DAS-59122-7× TC1507 with Mycogen Seeds/Dow 68467-6 Cry34Ab1/Cry35Ab1 + Cry1F AgroPioneer Hi- 29964-5 Bred/Dupont Herculex Xtra MON 88017 × MON 810 withCry1AB + Monsanto 524-552 Cry3Bb YieldGard VT Triple YieldGard VT PlusMIR 604 × Bt11 with mCry3A + Cry1Ab Syngenta 67979-8 Agrisure CB/RWAgrisure 3000GT Mon 89034 × Mon 88017 with Cry1A.105 + Monsanto 524-576Cry2Ab2 + Cry3Bb1 Genuity VT Triple PRO Bt11 × MIR 162 with Cry1Ab +Vip3Aa 20 Syngenta Seeds 67979-12 Agrisure 2100 Bt11 × MIR 162 × MIR 604with Cry1Ab + Syngenta Seeds 67979-13 Vip3Aa20 + mCry3A Agrisure 3100MON 89034 × TC1507 × MON 88017 × Monsanto Company 524-581 DAS-59122-7with Cry1A.105 + Cry2Ab2 + Mycogen Seeds/Dow 68467-7 Cry1F + Cry3Bb1 +Agro Cry34Ab1/Cry35Ab1 Genuity SmartStax SmartStax MON 89034 × TC1507 xMON 88017 × Monsanto Company 524-595 DAS-59122-7 Seed Blend MycogenSeeds/Dow 68467-16 Agro Genuity SmartStax RIB Complete SmartStax RefugeAdvanced; Refuge Advanced Powered by SmartStax Seed Blend of HerculexXtra + Herculex I Pioneer Hi- 29964-6 Bred/Dupont Optimum AcreMax1Insect Protection Seed Blend of Herculex RW + Non-Bt corn Pioneer Hi-29964-10 Bred/Dupont Optimum AcreMax RW (Cry1F × Cry34/35 × Cry1Ab) −seed blend Pioneer Hi- 29964-11 Bred/Dupont Optimum AcreMax Xtra (Cry1F× Cry1Ab) − seed blend Pioneer Hi- 29964-12 Bred/Dupont Optimum AcreMaxInsect Protection (Cry1F × mCry3A) Pioneer Hi- 29964-13 Bred/DupontOptimum Trisect (Cry1F × Cry34/35 × Cry1Ab × mCry3A) Pioneer Hi-29964-14 Bred/Dupont Optimum Intrasect Xtreme 59122 × MON 810 × MIR 604(Cry34/35 × Pioneer Hi- 29964-15 Cry1Ab × mCry3A) Bred/Dupont OptimumAcreMax Xtreme (Cry1F × Pioneer Hi- 29964-16 Cry34/35 × Cry1Ab × mCry3A)− seed Bred/Dupont blend Optimum AcreMax Xtreme (seed blend) MON 810 ×MIR 604 (Cry1Ab × mCry3A) Pioneer Hi- 29964-18 Bred/Dupont 1507 × MON810× MIR 162 (Cry1F × Pioneer Hi- 29964-19 Cry1Ab × Vip 3Aa20) Bred/DupontOptimum Intrasect Leptra 1507 × MIR 162 (Cry1F × Vip30Aa20) Pioneer Hi-29964-20 Bred/Dupont 4114 × MON 810 × MIR 604 (Cry34/35 × Pioneer Hi-29964-21 Cry1F × Cry1Ab × mCry3A) − seed blend Bred/Dupont 4114 × MON810 × MIR 604 (Cry34/35 × Pioneer Hi- 29964-22 Cry1F × Cry1Ab × mCry3A)Bred/Dupont 1507 × MON810 × MIR 604 (Cry1F × Pioneer Hi- 29964-23 Cry1Ab× mCry3A) − seed blend Bred/Dupont Optimum AcreMax Trisect 1507 × MON810× MIR 604 (Cry1F × Pioneer Hi- 29964-24 Cry1Ab × mCry3A) Bred/DupontOptimum Intrasect Trisect 4114 × MON 810 (Cry34/35 × Cry1F × Pioneer Hi-29964-25 Cry1Ab) Bred/Dupont 1507 × MON810 × MIR 162 (Cry1F × PioneerHi- 29964-26 Cry1Ab × Vip 3Aa20) − seed blend Bred/Dupont OptimumAcreMax Leptra SmartStax Intermediates (8 products) Monsanto 524-583,524-584, 524- 586, 524-587, 524- 588, 524-589, 524-590 MON 89034 × 1507(Cry1A.105 × Monsanto 524-585 Cry2Ab2 × Cry1F) Genuity Power-Core MON89034 (Cry1A.105 × Cry2Ab2) − Monsanto 524-597 seed blend Genuity VTDouble PRO RIB Complete MON 89034 × 88017 RIB Complete Monsanto 524-606(Cry1A.105 × Cry2Ab2 × Cry3Bb1) − seed Genuity VT Triple PRO blend RIBComplete MON 89034 × 1507 (Cry1A.105 × Monsanto 524-612 Cry2Ab2 × Cry1F)− seed blend Genuity PowerCore RIB Complete Bt11 × MIR162 × 1507 (Cry1Ab× Syngenta Seeds 67979-15 Vip3Aa20 × Cry1F) Agrisure Viptera 3220 RefugeRenew Bt11 × 59122-7 × MIR 604 × 1507 (Cry1Ab × Syngenta Seeds 67979-17Cry34/35 × mCry3A × Cry1F) Agrisure 3122 Bt11 × MIR162 × TC1507 (Cry1Ab× Syngenta Seeds 67979-19 Vip3Aa20 × Cry1F) − seed blend Agisure Viptera3220 (E-Z Refuge) (Refuge Advanced) Bt11 × DAS 59122-7 × MIR604 × TC1507Syngenta Seeds 67979-20 (Cry1Ab × Cry34/35 × mCry3A × Cry1F) − AgisureViptera 3122 seed blend (E-Z Refuge) (Refuge Advanced) Bt11 × MIR 162 ×MIR 604 × TC1507 × Syngenta Seeds 67979-23 5307 (Cry1Ab × Vip3Aa20 ×mCry3A × Agrisure Duracade Cry1F × eCry3.1Ab) (Refuge Renew) 5222 Bt11 ×MIR 604 × TC1507 × 5307 (Cry1Ab × Syngenta Seeds 67979-24 mCry3A × Cry1F× eCry3.1Ab) Agrisure Duracade (Refuge Renew) 5122 Bt11 × MIR 604 ×TC1507 × 5307 (Cry1Ab × Syngenta Seeds 67979-25 mCry3A × Cry1F ×eCry3.1Ab) − seed Agisure Duracade 5122 blend E-Z Refuge Bt11 × MIR 162× MIR 604 × TC1507 × Syngenta Seeds 67979-26 5307 (Cry1Ab × Vip3Aa20 ×mCry3A × Agisure Duracade 5222 Cry1F × eCry3.1Ab) − seed blend E-ZRefuge Bt11 × MIR 162 × MIR 604 × TC1507 × Syngenta Seeds 67979-27 5307(Cry1Ab × Vip3Aa20 x mCry3A × Agrisure Duracade Cry1F × eCry3.1Ab)(Refuge Renew) 5022 MIR604 × DAS-59122-7 × TC1507 Syngenta Seeds67979-29 (mCry3A × Cry34/35 x Cry1F) SmartStax Intermediates (8products) Mycogen Seeds/Dow 68467-8, 68467-9, Agro 68467-10, 68467-11,68467-13, 68467-14, 68467-15 MON 89034 × 1507 (Cry1A.105 × MycogenSeeds/Dow 68467-12 Cry2Ab2 × Cry1F) Agro PowerCore; PowerCore Enlist MON89034 × 1507 (Cry1A.105 × Mycogen Seeds/Dow 68467-21 Cry2Ab2 × Cry1F) −seed blend Agro PowerCore Refuge Advanced; Refuge Advanced Powered byPowerCore 1507 × MON 810 Pioneer Hi- 29964-7 Bred/Dupont OptimumIntrasect 59122 × 1507 × MON 810 Pioneer Hi- 29964-8 Bred/Dupont 59122 ×MON 810 Pioneer Hi- 29964-9 Bred/Dupont Cotton Cry1Ac Cotton Monsanto524-478 BollGard Cry1Ac and Cry2Ab2 in Event 15985 Monsanto 524-522Cotton PC Codes 006445, 006487 BollGard II Bt cotton Event MON531 withCry1Ac Monsanto 524-555 (breeding nursery use only) Bt cotton EventMON15947 with Cry2Ab2 Monsanto 524-556 (breeding nursery use only)COT102 × MON 15985 (Vip3Aa19 × Monsanto 524-613 Cry1Ac × Cry2Ab2)Bollgard III Cry1F and Cry1Ac (Events DAS-21023-5 × Mycogen Seeds/Dow68467-3 DAS-24236-5) Cotton PC Codes 006512, Agro 006513 WidestrikeEvent 3006-210-23 (Cry1Ac) Mycogen Seeds/Dow 68467-17 Agro Event281-24-236 (Cry1F) Mycogen Seeds/Dow 68467-18 Agro WideStrike × COT102(Cry1F × Cry1Ac × Mycogen Seeds/Dow 68467-19 Vip3Aa19) Agro WideStrike 3Vip3Aa19 and FLCry1Ab (Events Syngenta Seeds 67979-9 Cot102 × Cot67B)Cotton PC Codes 016484, (Formally VipCot) 016486 OECD Unique IdentifierSYN- IR102-7 X SYN-IR67B-1 COT102 (Vip3Aa19) Syngenta Seeds 67979-18COT67B (FLCry1Ab) Syngenta Seeds 67979-21 T304-40 (Cry1Ab) BayerCropScience 264-1094 GHB119 (Cry2Ae) Bayer CropScience 264-1095 T304-40× GHB119 (Cry1Ab × Cry2Ae) Bayer CropScience 264-1096 OECD UniqueIdentifier: BCS-GHØØ4-7 × TwinLink BCS-GHØØ5-8 Soybean Cry1Ac in Event87701 Soybean PC Code Monsanto 524-594 006532 OECD Unique IdentifierInacta Cry1A.105 and Cry2Ab2 in Event 87751 Monsanto 524-619 Soybean PCCodes 006614, 006615 OECD Unique Identifier MON-87751-7 Cry1Ac × Cry1Fin Event DAS 81419 Mycogen Seeds/Dow 68467-20 Soybean PC Codes 006527,006528 OECD Agro Unique Identifier DAS 81419 (Cry1Ac × Cry1F)

In some embodiments, any one or more of the pesticides set forth hereinmay be utilized with any one or more of the microbes of the disclosureand can be applied to plants or parts thereof, including seeds.

Herbicides

As aforementioned, agricultural compositions of the disclosure, whichmay comprise any microbe taught herein, are sometimes combined with oneor more herbicides.

Compositions comprising bacteria or bacterial populations producedaccording to methods described herein and/or having characteristics asdescribed herein may further include one or more herbicides. In someembodiments, herbicidal compositions are applied to the plants and/orplant parts. In some embodiments, herbicidal compositions may beincluded in the compositions set forth herein, and can be applied to aplant(s) or a part(s) thereof simultaneously or in succession, withother compounds.

Herbicides include 2,4-D, 2,4-DB, acetochlor, acifluorfen, alachlor,ametryn, atrazine, aminopyralid, benefin, bensulfuron, bensulide,bentazon, bicyclopyrone, bromacil, bromoxynil, butylate, carfentrazone,chlorimuron, chlorsulfuron, clethodim, clomazone, clopyralid,cloransulam, cycloate, DCPA, desmedipham, dicamba, dichlobenil,diclofop, diclosulam, diflufenzopyr, dimethenamid, diquat, diuron, DSMA,endothall, EPTC, ethalfluralin, ethofumesate, fenoxaprop, fluazifop-P,flucarbzone, flufenacet, flumetsulam, flumiclorac, flumioxazin,fluometuron, fluroxypyr, fomesafen, foramsulfuron, glufosinate,glyphosate, halosulfuron, hexazinone, imazamethabenz, imazamox,imazapic, imazaquin, imazethapyr, isoxaflutole, lactofen, linuron, MCPA,MCPB, mesotrione, metolachlor-s, metribuzin, indaziflam, metsulfuron,molinate, MSMA, napropamide, naptalam, nicosulfuron, norflurazon,oryzalin, oxadiazon, oxyfluorfen, paraquat, pelargonic acid,pendimethalin, phenmedipham, picloram, primisulfuron, prodiamine,prometryn, pronamide, propanil, prosulfuron, pyrazon, pyrithioac,quinclorac, quizalofop, rimsulfuron, S-metolachlor, sethoxydim, siduron,simazine, sulfentrazone, sulfometuron, sulfosulfuron, tebuthiuron,tembotrione, terbacil, thiazopyr, thifensulfuron, thiobencarb,topramezone, tralkoxydim, triallate, triasulfuron, tribenuron,triclopyr, trifluralin, and triflusulfuron.

In some embodiments, any one or more of the herbicides set forth hereinmay be utilized with any one or more of the plants or parts thereof setforth herein.

Herbicidal products may include CORVUS, BALANCE FLEXX, CAPRENO, DIFLEXX,LIBERTY, LAUDIS, AUTUMN SUPER, and DIFLEXX DUO.

In some embodiments, any one or more of the herbicides set forth in thebelow Table 12 may be utilized with any one or more of the microbestaught herein, and can be applied to any one or more of the plants orparts thereof set forth herein.

TABLE 12 List of exemplary herbicides, which can be combined withmicrobes of the disclosure Herbicide Site of Action Group NumberChemical Family Herbicide ACCase 1 Cyclohexanediones Sethoxydim (Poast,inhibitors Poast Plus) Clethodim (Select, Select Max, Arrow)Aryloxyphenoxypropionates Fluazifop (Fusilade DX, component in Fusion)Fenoxaprop (Puma, component in Fusion) Quizalofop (Assure II, Targa)Phenylpyrazolins Pinoxaden (Axial XL) ALS inhibitors 2 ImidazolinonesImazethapyr (Pursuit) Imazamox (Raptor) Sulfonylureas Chlorimuron(Classic) Halosulfuron (Permit, Sandea) Iodosulfuron (component inAutumn Super) Mesosulfuron (Osprey) Nicosulfuron (Accent Q)Primisulfuron (Beacon) Prosulfuron (Peak) Rimsulfuron (Matrix, Resolve)Thifensulfuron (Harmony) Tribenuron (Express) Triflusulfuron (UpBeet)Triazolopyrimidine Flumetsulam (Python) Cloransulam-methyl (FirstRate)Pyroxsulam (PowerFlex HL) Florasulam (component in Quelex)Sulfonylaminocarbonyltriazolinones Propoxycarbazone (Olympus)Thiencarbazone-methyl (component in Capreno) Microtubule 3Dinitroanilines Trifluralin (many inhibitors (root names) inhibitors)Ethalfluralin (Sonalan) Pendimethalin (Prowl/Prowl H₂O) BenzamidePronamide (Kerb) Synthetic auxins 4 Arylpicolinate Halauxifen (Elevore,component in Quelex) Phenoxy acetic acids 2,4-D (Enlist One, others)2,4-DB (Butyrac 200, Butoxone 200) MCPA Benzoic acids Dicamba (Banvel,Clarity, DiFlexx, Eugenia, XtendiMax; component in Status) PyridinesClopyralid (Stinger) Fluroxypyr (Starane Ultra) Photosystem II 5Triazines Atrazine inhibitors Simazine (Princep, Sim- Trol) TriazinoneMetribuzin (Metribuzin, others) Hexazinone (Velpar) Phenyl-carbamatesDesmedipham (Betenex) Phenmedipham (component in Betamix) UracilsTerbacil (Sinbar) 6 Benzothiadiazoles Bentazon (Basagran, others)Nitriles Bromoxynil (Buctril, Moxy, others) 7 Phenylureas Linuron(Lorox, Linex) Lipid synthesis 8 Thiocarbamates EPTC (Eptam) inhibitorEPSPS inhibitor 9 Organophosphorus Glyphosate Glutamine 10Organophosphorus Glufosinate (Liberty, synthetase Rely) inhibitorDiterpene 13 Isoxazolidinone Clomazone (Command) biosynthesis inhibitor(bleaching) Protoporphyrinogen 14 Diphenylether Acifluorfen (Ultraoxidase Blazer) inhibitors (PPO) Fomesafen (Flexstar, Reflex) Lactofen(Cobra, Phoenix) N-phenylphthalimide Flumiclorac (Resource) Flumioxazin(Valor, Valor EZ, Rowel) Aryl triazolinone Sulfentrazone (Authority,Spartan) Carfentrazone (Aim) Fluthiacet-methyl (Cadet) PyrazolesPyraflufen-ethyl (Vida) Pyrimidinedione Saflufenacil (Sharpen)Long-chain fatty 15 Acetamides Acetochlor (Harness, acid inhibitorsSurpass NXT, Breakfree NXT, Warrant) Dimethenamid-P (Outlook)Metolachlor (Parallel) Pyroxasulfone (Zidua, Zidua SC) s-metolachlor(Dual Magnum, Dual II Magnum, Cinch) Flufenacet (Define) Specific site16 Benzofuranes Ethofumesate (Nortron) unknown Auxin transport 19Semicarbazone diflufenzopyr inhibitor (component in Status) PhotosystemI 22 Bipyridiliums Paraquat (Gramoxone, inhibitors Parazone) Diquat(Reglone) 4-HPPD 27 Isoxazole Isoxaflutole (Balance inhibitors PyrazoleFlexx) (bleaching) Pyrazolone Pyrasulfotole Triketone (component inHuskie) Topramezone (Armezon/Impact) Bicyclopyrone (component in Acuron)Mesotrione (Callisto) Tembotrione (Laudis)

Fungicides

As aforementioned, agricultural compositions of the disclosure, whichmay comprise any microbe taught herein, are sometimes combined with oneor more fungicides.

Compositions comprising bacteria or bacterial populations producedaccording to methods described herein and/or having characteristics asdescribed herein may further include one or more fungicides. In someembodiments, fungicidal compositions may be included in the compositionsset forth herein, and can be applied to a plant(s) or a part(s) thereofsimultaneously or in succession, with other compounds. The fungicidesinclude azoxystrobin, captan, carboxin, ethaboxam, fludioxonil,mefenoxam, fludioxonil, thiabendazole, thiabendaz, ipconazole, mancozeb,cyazofamid, zoxamide, metalaxyl, PCNB, metaconazole, pyraclostrobin,Bacillus subtilis strain QST 713, sedaxane, thiamethoxam, fludioxonil,thiram, tolclofos-methyl, trifloxystrobin, Bacillus subtilis strain MBI600, pyraclostrobin, fluoxastrobin, Bacillus pumilus strain QST 2808,chlorothalonil, copper, flutriafol, fluxapyroxad, mancozeb, gludioxonil,penthiopyrad, triazole, propiconaozole, prothioconazole, tebuconazole,fluoxastrobin, pyraclostrobin, picoxystrobin, qols, tetraconazole,trifloxystrobin, cyproconazole, flutriafol, SDHI, EBDCs, sedaxane, MAXIMQUATTRO (gludioxonil, mefenoxam, azoxystrobin, and thiabendaz), RAXIL(tebuconazole, prothioconazole, metalaxyl, and ethoxylated tallow alkylamines), and benzovindiflupyr.

In some embodiments, any one or more of the fungicides set forth hereinmay be utilized with any one or more of the plants or parts thereof setforth herein.

Nematicides

As aforementioned, agricultural compositions of the disclosure, whichmay comprise any microbe taught herein, are sometimes combined with oneor more nematicides.

Compositions comprising bacteria or bacterial populations producedaccording to methods described herein and/or having characteristics asdescribed herein may further include one or more nematicide. In someembodiments, nematicidal compositions may be included in thecompositions set forth herein, and can be applied to a plant(s) or apart(s) thereof simultaneously or in succession, with other compounds.The nematicides may be selected from D-D, 1,3-dichloropropene, ethylenedibromide, 1,2-dibromo-3-chloropropane, methyl bromide, chloropicrin,metam sodium, dazomet, methylisothiocyanate, sodium tetrathiocarbonate,aldicarb, aldoxycarb, carbofuran, oxamyl, ethoprop, fenamiphos,cadusafos, fosthiazate, terbufos, fensulfothion, phorate, DiTera,clandosan, sincocin, methyl iodide, propargyl bromide,2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine (DMDP), any one or more ofthe avermectins, sodium azide, furfural, Bacillus firmus, abamectrin,thiamethoxam, fludioxonil, clothiandin, salicylic acid, andbenzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester.

In some embodiments, any one or more of the nematicides set forth hereinmay be utilized with any one or more of the plants or parts thereof setforth herein.

In some embodiments, any one or more of the nematicides, fungicides,herbicides, insecticides, and/or pesticides set forth herein may beutilized with any one or more of the plants or parts thereof set forthherein.

Fertilizers, Nitrogen Stabilizers, and Urease Inhibitors

As aforementioned, agricultural compositions of the disclosure, whichmay comprise any microbe taught herein, are sometimes combined with oneor more of a: fertilizer, nitrogen stabilizer, or urease inhibitor.

In some embodiments, fertilizers are used in combination with themethods and bacteria of the present disclosure. Fertilizers includeanhydrous ammonia, urea, ammonium nitrate, and urea-ammonium nitrate(UAN) compositions, among many others. In some embodiments, pop-upfertilization and/or starter fertilization is used in combination withthe methods and bacteria of the present disclosure.

In some embodiments, nitrogen stabilizers are used in combination withthe methods and bacteria of the present disclosure. Nitrogen stabilizersinclude nitrapyrin, 2-chloro-6-(trichloromethyl) pyridine, N-SERVE 24,INSTINCT, dicyandiamide (DCD).

In some embodiments, urease inhibitors are used in combination with themethods and bacteria of the present disclosure. Urease inhibitorsinclude N-(n-butyl)-thiophosphoric triamide (NBPT), AGROTAIN, AGROTAINPLUS, and AGROTAIN PLUS SC. Further, the disclosure contemplatesutilization of AGROTAIN ADVANCED 1.0, AGROTAIN DRI-MAXX, and AGROTAINULTRA.

Further, stabilized forms of fertilizer can be used. For example, astabilized form of fertilizer is SUPER U, containing 46% nitrogen in astabilized, urea-based granule, SUPERU contains urease and nitrificationinhibitors to guard from denitrification, leaching, and volatilization.Stabilized and targeted foliar fertilizer such as NITAMIN may also beused herein.

Pop-up fertilizers are commonly used in corn fields. Pop-upfertilization comprises applying a few pounds of nutrients with the seedat planting. Pop-up fertilization is used to increase seedling vigor.

Slow- or controlled-release fertilizer that may be used herein entails:A fertilizer containing a plant nutrient in a form which delays itsavailability for plant uptake and use after application, or whichextends its availability to the plant significantly longer than areference ‘rapidly available nutrient fertilizer’ such as ammoniumnitrate or urea, ammonium phosphate or potassium chloride. Such delay ofinitial availability or extended time of continued availability mayoccur by a variety of mechanisms. These include controlled watersolubility of the material by semi-permeable coatings, occlusion,protein materials, or other chemical forms, by slow hydrolysis ofwater-soluble low molecular weight compounds, or by other unknown means.

Stabilized nitrogen fertilizer that may be used herein entails: Afertilizer to which a nitrogen stabilizer has been added. A nitrogenstabilizer is a substance added to a fertilizer which extends the timethe nitrogen component of the fertilizer remains in the soil in theurea-N or ammoniacal-N form.

Nitrification inhibitor that may be used herein entails: A substancethat inhibits the biological oxidation of ammoniacal-N to nitrate-N.Some examples include: (1) 2-chloro-6-(trichloromethyl-pyridine), commonname Nitrapyrin, manufactured by Dow Chemical; (2)4-amino-1,2,4-6-triazole-HCl, common name ATC, manufactured by IshihadaIndustries; (3) 2,4-diamino-6-trichloro-methyltriazine, common nameCI-1580, manufactured by American Cyanamid; (4) Dicyandiamide, commonname DCD, manufactured by Showa Denko; (5) Thiourea, common name TU,manufactured by Nitto Ryuso; (6) 1-mercapto-1,2,4-triazole, common nameMT, manufactured by Nippon; (7) 2-amino-4-chloro-6-methyl-pyramidine,common name AM, manufactured by Mitsui Toatsu; (8) 3,4-dimethylpyrazolephosphate (DMPP), from BASF; (9) 1-amide-2-thiourea (ASU), from NittoChemical Ind.; (10) Ammoniumthiosulphate (ATS); (11) 1H-1,2,4-triazole(HPLC); (12) 5-ethylene oxide-3-trichloro-methlyl 1,2,4-thiodiazole(Terrazole), from Olin Mathieson; (13) 3-methylpyrazole (3-MP); (14)1-carbamoyle-3-methyl-pyrazole (CMP); (15) Neem; and (16) DMPP.

Urease inhibitor that may be used herein entails: A substance thatinhibits hydrolytic action on urea by the enzyme urease. Thousands ofchemicals have been evaluated as soil urease inhibitors (Kiss andSimihaian, 2002). However, only a few of the many compounds tested meetthe necessary requirements of being non toxic, effective at lowconcentration, stable, and compatible with urea (solid and solutions),degradable in the soil and inexpensive. They can be classified accordingto their structures and their assumed interaction with the enzyme urease(Watson, 2000, 2005). Four main classes of urease inhibitors have beenproposed: (a) reagents which interact with the sulphydryl groups(sulphydryl reagents), (b) hydroxamates, (c) agricultural cropprotection chemicals, and (d) structural analogues of urea and relatedcompounds. N-(n-Butyl) thiophosphoric triamide (NBPT),phenylphosphorodiamidate (PPD/PPDA), and hydroquinone are probably themost thoroughly studied urease inhibitors (Kiss and Simihaian, 2002).Research and practical testing has also been carried out withN-(2-nitrophenyl) phosphoric acid triamide (2-NPT) and ammoniumthiosulphate (ATS). The organo-phosphorus compounds are structuralanalogues of urea and are some of the most effective inhibitors ofurease activity, blocking the active site of the enzyme (Watson, 2005).

Insecticidal Seed Treatments (ISTs) for Corn

Corn seed treatments normally target three spectrums of pests:nematodes, fungal seedling diseases, and insects.

Insecticide seed treatments are usually the main component of a seedtreatment package. Most corn seed available today comes with a basepackage that includes a fungicide and insecticide. In some aspects, theinsecticide options for seed treatments include PONCHO (clothianidin),CRUISER/CRUISER EXTREME (thiamethoxam) and GAUCHO (Imidacloprid). Allthree of these products are neonicotinoid chemistries. CRUISER andPONCHO at the 250 (0.25 mg AI/seed) rate are some of the most commonbase options available for corn. In some aspects, the insecticideoptions for treatments include CRUISER 250 thiamethoxam, CRUISER 250(thiamethoxam) plus LUMIVIA (chlorantraniliprole), CRUISER 500(thiamethoxam), and PONCHO VOTIVO 1250 (Clothianidin & Bacillus firmus1-1582).

Pioneer's base insecticide seed treatment package consists of CRUISER250 with PONCHO/VOTIVO 1250 also available. VOTIVO is a biological agentthat protects against nematodes.

Monsanto's products including corn, soybeans, and cotton fall under theACCELERON treatment umbrella. Dekalb corn seed comes standard withPONCHO 250. Producers also have the option to upgrade to PONCHO/VOTIVO,with PONCHO applied at the 500 rate.

Agrisure, Golden Harvest and Garst have a base package with a fungicideand CRUISER 250. AVICTA complete corn is also available; this includesCRUISER 500, fungicide, and nematode protection. CRUISER EXTREME isanother option available as a seed treatment package, however; theamounts of CRUISER are the same as the conventional CRUISER seedtreatment, i.e. 250, 500, or 1250.

Another option is to buy the minimum insecticide treatment available,and have a dealer treat the seed downstream.

Commercially available ISTs for corn are listed in the below Table 13and can be combined with one or more of the microbes taught herein.

TABLE 13 List of exemplary seed treatments, including ISTs, which can becombined with microbes of the disclosure Treatment Type ActiveIngredient(s) Product Trade Name Crop F azoxystrobin DYNASTY Corn,Soybean PROTÉGÉ FL Corn F Bacillus pumilus YIELD SHIELD Corn, Soybean FBacillus subtilis HISTICK N/T Soybean VAULT HP Corn, Soybean F CaptanCAPTAN 400 Corn, Soybean CAPTAN 400-C Corny Soybean F Fludioxonil MAXIM4FS Corn, Soybean F Hydrogen peroxide OXIDATE Soybean STOROX Soybean Fipconazole ACCELERON DC-509 Corn RANCONA 3.8 FS Corn, Soybean VORTEXCorn F mancozeb BONIDE MANCOZEB w/Zinc Corn Concentrate DITHANE 75DFRAINSHIELD Corn DITHANE DF RAINSHIELD Corn DITHANE F45 RAINSHIELD CornDITHANE M45 Corn LESCO 4 FLOWABLE Corn MANCOZEB PENNCOZEB 4FL FLOWABLECorn PENNCOZEB 75DF DRY Corn FLOWABLE PENNCOZEB 80WP Corn F mefenoxamAPRON XL Corn, Soybean F metalaxyl ACCELERON DC-309 Corn ACCELERONDX-309 Corn, Soybean ACQUIRE Corn, Soybean AGRI STAR METALAXYL 265 Corn,Soybean ST ALLEGIANCE DRY Corn, Soybean ALLEGIANCE FL Corn, SoybeanBELMONT 2.7 FS Corn, Soybean DYNA-SHIELD METALAXYL Corn, Soybean SEBRING2.65 ST Corn, Soybean SEBRING 318 FS Corn, Soybean SEBRING 480 FS Corn,Soybean VIREO MEC Soybean F pyraclostrobin ACCELERON DX-109 SoybeanSTAMINA Corn F Streptomyces MYCOSTOP Corn, Soybean griseoviridis FStreptomyces lydicus ACTINOGROW ST Corn, Soybean F tebuconazole AMTIDETEBU 3.6F Corn SATIVA 309 FS Corn SATIVA 318 FS Corn TEBUSHA 3.6FL CornTEBUZOL 3.6F Corn F thiabendazole MERTECT 340-F Soybean F thiram 42-STHIRAM Corn, Soybean FLOWSAN Corn, Soybean SIGNET 480 FS Corn, Soybean FTrichoderma T-22 HC Corn, Soybean harzianum Rifai F trifloxystrobinACCELERON DX-709 Corn TRILEX FLOWABLE Corn, soybean I chlorpyrifosLORSBAN 50W in water soluble Corn packets I clothianidin ACCELERONIC-609 Corn NIPSIT INSIDE Corn, Soybean PONCHO 600 Corn I imidaclopridACCELERON IX-409 Corn AGRI STAR MACHO 600 ST Corn, Soybean AGRISOLUTIONSNITRO Corn, Soybean SHIELD ATTENDANT 600 Corn, Soybean AXCESS Corn,Soybean COURAZE 2F Soybean DYNA-SHIELD Corn, Soybean IMIDACLOPRID 5GAUCHO 480 FLOWABLE Corn, Soybean GAUCHO 600 FLOWABLE Corn, SoybeanGAUCHO SB FLOWABLE Corn, Soybean NUPRID 4.6F PRO Soybean SENATOR 600 FSCorn, Soybean I thiamethoxam CRUISER 5FS Corn, Soybean N abamectinAVICTA 500 FS Corn, Soybean N Bacillus firmus VOTIVO FS Soybean Pcytokinin SOIL X-CYTO Soybean X-CYTE Soybean P harpin alpha betaACCELERON HX-209 Corn, Soybean protein N-HIBIT GOLD CST Corn, SoybeanN-HIBIT HX-209 Corn, Soybean P indole butyric acid KICKSTAND PGR Corn,Soybean I, N thiamethoxam, AVICTA DUO CORN Corn abamectin AVICTA DUO 250I, F clothianidin, PONCHO VOTIVO Corn, Soybean Bacillus firmus F, Fcarboxin, captan ENHANCE Soybean I, F permethrin, carboxin KERNEL GUARDSUPREME Corn, Soybean F, F carboxin, thiram VITAFLO 280 Corn, Soybean F,F mefenoxam, fludioxonil MAXIM XL Corn, Soybean WARDEN RTA Soybean APRONMAXX RFC APRON MAXX RTA + MOLY APRON MAXX RTA I, F imidacloprid,metalaxyl AGRISOLUTIONS CONCUR Corn F, F metalaxyl, ipconazole RANCONASUMMIT Soybean RANCONA XXTRA F, F thiram, metalaxylPROTECTOR-L-ALLEGIANCE Soybean F, F trifloxystrobin, TRILEX AL Soybeanmetalaxyl TRILEX 2000 P, P, P cytokinin, gibberellic STIMULATE YIELDCorn, Soybean acid, indole butyric acid ENHANCER ASCEND F, F, Imefenoxam, CRUISERMAXX PLUS Soybean fludioxonil, thiamethoxam F, F, Fcaptan, carboxin, BEAN GUARD/ALLEGIANCE Soybean metalaxyl F, F, Icaptan, carboxin, ENHANCE AW Soybean imidacloprid F, F, I carboxin,LATITUDE Corn, Soybean metalaxyl, imidacloprid F, F, F metalaxyl,STAMINA F3 HL Corn pyraclostrobin, triticonazole F, F, F, Iazoxystrobin, CRUISER EXTREME Corn fludioxonil, mefenoxam, thiamethoxamF, F, F, F, F azoxystrobin, MAXIM QUATTRO Corn fludioxonil, mefenoxam,thiabendazole I Chlorantraniliprole LUMIVIA Corn F = Fungicide; I =Insecticide; N = Nematicide; P = Plant Growth Regulator

Application of Bacterial Populations on Crops

The composition of the bacteria or bacterial population described hereincan be applied in furrow, in talc, or as seed treatment. The compositioncan be applied to a seed package in bulk, mini bulk, in a bag, or intalc.

The planter can plant the treated seed and grows the crop according toconventional ways, twin row, or ways that do not require tilling. Theseeds can be distributed using a control hopper or an individual hopper.Seeds can also be distributed using pressurized air or manually. Seedplacement can be performed using variable rate technologies.Additionally, application of the bacteria or bacterial populationdescribed herein may be applied using variable rate technologies. Insome examples, the bacteria can be applied to seeds of corn, soybean,canola, sorghum, potato, rice, vegetables, cereals, pseudocereals, andoilseeds. Examples of cereals may include barley, fonio, oats, palmer'sgrass, rye, pearl millet, sorghum, spelt, teff, triticale, and wheat.Examples of pseudocereals may include breadnut, buckwheat, cattail,chia, flax, grain amaranth, hanza, quinoa, and sesame. In some examples,seeds can be genetically modified organisms (GMO), non-GMO, organic orconventional.

Additives such as micro-fertilizer, PGR, herbicide, insecticide, andfungicide can be used additionally to treat the crops. Examples ofadditives include crop protectants such as insecticides, nematicides,fungicide, enhancement agents such as colorants, polymers, pelleting,priming, and disinfectants, and other agents such as inoculant, PGR,softener, and micronutrients. PGRs can be natural or synthetic planthormones that affect root growth, flowering, or stem elongation. PGRscan include auxins, gibberellins, cytokinins, ethylene, and abscisicacid (ABA).

The composition can be applied in furrow in combination with liquidfertilizer. In some examples, the liquid fertilizer may be held intanks. NPK fertilizers contain macronutrients of sodium, phosphorous,and potassium.

The composition may improve plant traits, such as promoting plantgrowth, maintaining high chlorophyll content in leaves, increasing fruitor seed numbers, and increasing fruit or seed unit weight. Methods ofthe present disclosure may be employed to introduce or improve one ormore of a variety of desirable traits. Examples of traits that mayintroduced or improved include: root biomass, root length, height, shootlength, leaf number, water use efficiency, overall biomass, yield, fruitsize, grain size, photosynthesis rate, tolerance to drought, heattolerance, salt tolerance, tolerance to low nitrogen stress, nitrogenuse efficiency, resistance to nematode stress, resistance to a fungalpathogen, resistance to a bacterial pathogen, resistance to a viralpathogen, level of a metabolite, modulation in level of a metabolite,proteome expression. The desirable traits, including height, overallbiomass, root and/or shoot biomass, seed germination, seedling survival,photosynthetic efficiency, transpiration rate, seed/fruit number ormass, plant grain or fruit yield, leaf chlorophyll content,photosynthetic rate, root length, or any combination thereof, can beused to measure growth, and compared with the growth rate of referenceagricultural plants (e.g., plants without the introduced and/or improvedtraits) grown under identical conditions. In some examples, thedesirable traits, including height, overall biomass, root and/or shootbiomass, seed germination, seedling survival, photosynthetic efficiency,transpiration rate, seed/fruit number or mass, plant grain or fruityield, leaf chlorophyll content, photosynthetic rate, root length, orany combination thereof, can be used to measure growth, and comparedwith the growth rate of reference agricultural plants (e.g., plantswithout the introduced and/or improved traits) grown under similarconditions.

An agronomic trait to a host plant may include, but is not limited to,the following: altered oil content, altered protein content, alteredseed carbohydrate composition, altered seed oil composition, and alteredseed protein composition, chemical tolerance, cold tolerance, delayedsenescence, disease resistance, drought tolerance, ear weight, growthimprovement, health e4nhancement, heat tolerance, herbicide tolerance,herbivore resistance improved nitrogen fixation, improved nitrogenutilization, improved root architecture, improved water use efficiency,increased biomass, increased root length, increased seed weight,increased shoot length, increased yield, increased yield underwater-limited conditions, kernel mass, kernel moisture content, metaltolerance, number of ears, number of kernels per ear, number of pods,nutrition enhancement, pathogen resistance, pest resistance,photosynthetic capability improvement, salinity tolerance, stay-green,vigor improvement, increased dry weight of mature seeds, increased freshweight of mature seeds, increased number of mature seeds per plant,increased chlorophyll content, increased number of pods per plant,increased length of pods per plant, reduced number of wilted leaves perplant, reduced number of severely wilted leaves per plant, and increasednumber of non-wilted leaves per plant, a detectable modulation in thelevel of a metabolite, a detectable modulation in the level of atranscript, and a detectable modulation in the proteome, compared to anisoline plant grown from a seed without said seed treatment formulation.

In some cases, plants are inoculated with bacteria or bacterialpopulations that are isolated from the same species of plant as theplant element of the inoculated plant. For example, an bacteria orbacterial population that is normally found in one variety of Zea mays(corn) is associated with a plant element of a plant of another varietyof Zea mays that in its natural state lacks said bacteria and bacterialpopulations. In one embodiment, the bacteria and bacterial populationsis derived from a plant of a related species of plant as the plantelement of the inoculated plant. For example, an bacteria and bacterialpopulations that is normally found in Zea diploperennis Iltis et al.,(diploperennial teosinte) is applied to a Zea mays (corn), or viceversa. In some cases, plants are inoculated with bacteria and bacterialpopulations that are heterologous to the plant element of the inoculatedplant. In one embodiment, the bacteria and bacterial populations isderived from a plant of another species. For example, an bacteria andbacterial populations that is normally found in dicots is applied to amonocot plant (e.g., inoculating corn with a soybean-derived bacteriaand bacterial populations), or vice versa. In other cases, the bacteriaand bacterial populations to be inoculated onto a plant is derived froma related species of the plant that is being inoculated. In oneembodiment, the bacteria and bacterial populations is derived from arelated taxon, for example, from a related species. The plant of anotherspecies can be an agricultural plant. In another embodiment, thebacteria and bacterial populations is part of a designed compositioninoculated into any host plant element.

In some examples, the bacteria or bacterial population is exogenouswherein the bacteria and bacterial population is isolated from adifferent plant than the inoculated plant. For example, in oneembodiment, the bacteria or bacterial population can be isolated from adifferent plant of the same species as the inoculated plant. In somecases, the bacteria or bacterial population can be isolated from aspecies related to the inoculated plant.

In some examples, the bacteria and bacterial populations describedherein are capable of moving from one tissue type to another. Forexample, the present disclosure's detection and isolation of bacteriaand bacterial populations within the mature tissues of plants aftercoating on the exterior of a seed demonstrates their ability to movefrom seed exterior into the vegetative tissues of a maturing plant.Therefore, in one embodiment, the population of bacteria and bacterialpopulations is capable of moving from the seed exterior into thevegetative tissues of a plant. In one embodiment, the bacteria andbacterial populations that is coated onto the seed of a plant iscapable, upon germination of the seed into a vegetative state, oflocalizing to a different tissue of the plant. For example, bacteria andbacterial populations can be capable of localizing to any one of thetissues in the plant, including: the root, adventitious root, seminal 5root, root hair, shoot, leaf, flower, bud, tassel, meristem, pollen,pistil, ovaries, stamen, fruit, stolon, rhizome, nodule, tuber,trichome, guard cells, hydathode, petal, sepal, glume, rachis, vascularcambium, phloem, and xylem. In one embodiment, the bacteria andbacterial populations is capable of localizing to the root and/or theroot hair of the plant. In another embodiment, the bacteria andbacterial populations is capable of localizing to the photosynthetictissues, for example, leaves and shoots of the plant. In other cases,the bacteria and bacterial populations is localized to the vasculartissues of the plant, for example, in the xylem and phloem. In stillanother embodiment, the bacteria and bacterial populations is capable oflocalizing to the reproductive tissues (flower, pollen, pistil, ovaries,stamen, fruit) of the plant. In another embodiment, the bacteria andbacterial populations is capable of localizing to the root, shoots,leaves and reproductive tissues of the plant. In still anotherembodiment, the bacteria and bacterial populations colonizes a fruit orseed tissue of the plant. In still another embodiment, the bacteria andbacterial populations is able to colonize the plant such that it ispresent in the surface of the plant (i.e., its presence is detectablypresent on the plant exterior, or the episphere of the plant). In stillother embodiments, the bacteria and bacterial populations is capable oflocalizing to substantially all, or all, tissues of the plant. Incertain embodiments, the bacteria and bacterial populations is notlocalized to the root of a plant. In other cases, the bacteria andbacterial populations is not localized to the photosynthetic tissues ofthe plant.

The effectiveness of the compositions can also be assessed by measuringthe relative maturity of the crop or the crop heating unit (CHU). Forexample, the bacterial population can be applied to corn, and corngrowth can be assessed according to the relative maturity of the cornkernel or the time at which the corn kernel is at maximum weight. Thecrop heating unit (CHU) can also be used to predict the maturation ofthe corn crop. The CHU determines the amount of heat accumulation bymeasuring the daily maximum temperatures on crop growth.

In examples, bacterial may localize to any one of the tissues in theplant, including: the root, adventitious root, seminal root, root hair,shoot, leaf, flower, bud tassel, meristem, pollen, pistil, ovaries,stamen, fruit, stolon, rhizome, nodule, tuber, trichome, guard cells,hydathode, petal, sepal, glume, rachis, vascular cambium, phloem, andxylem. In another embodiment, the bacteria or bacterial population iscapable of localizing to the photosynthetic tissues, for example, leavesand shoots of the plant. In other cases, the bacteria and bacterialpopulations is localized to the vascular tissues of the plant, forexample, in the xylem and phloem. In another embodiment, the bacteria orbacterial population is capable of localizing to reproductive tissues(flower, pollen, pistil, ovaries, stamen, or fruit) of the plant. Inanother embodiment, the bacteria and bacterial populations is capable oflocalizing to the root, shoots, leaves and reproductive tissues of theplant. In another embodiment, the bacteria or bacterial populationcolonizes a fruit or seed tissue of the plant. In still anotherembodiment, the bacteria or bacterial population is able to colonize theplant such that it is present in the surface of the plant. In anotherembodiment, the bacteria or bacterial population is capable oflocalizing to substantially all, or all, tissues of the plant. Incertain embodiments, the bacteria or bacterial population is notlocalized to the root of a plant. In other cases, the bacteria andbacterial populations is not localized to the photosynthetic tissues ofthe plant.

The effectiveness of the bacterial compositions applied to crops can beassessed by measuring various features of crop growth including, but notlimited to, planting rate, seeding vigor, root strength, droughttolerance, plant height, dry down, and test weight.

Plant Species

The methods and bacteria described herein are suitable for any of avariety of plants, such as plants in the genera Hordeum, Oryza, Zea, andTriticeae. Other non-limiting examples of suitable plants includemosses, lichens, and algae. In some cases, the plants have economic,social and/or environmental value, such as food crops, fiber crops, oilcrops, plants in the forestry or pulp and paper industries, feedstockfor biofuel production and/or ornamental plants. In some examples,plants may be used to produce economically valuable products such as agrain, a flour, a starch, a syrup, a meal, an oil, a film, a packaging,a nutraceutical product, a pulp, an animal feed, a fish fodder, a bulkmaterial for industrial chemicals, a cereal product, a processedhuman-food product, a sugar, an alcohol, and/or a protein. Non-limitingexamples of crop plants include maize, rice, wheat, barley, sorghum,millet, oats, rye triticale, buckwheat, sweet corn, sugar cane, onions,tomatoes, strawberries, and asparagus. In some embodiments, the methodsand bacteria described herein are suitable for any of a variety oftransgenic plants, non-transgenic plants, and hybrid plants thereof.

In some examples, plants that may be obtained or improved using themethods and composition disclosed herein may include plants that areimportant or interesting for agriculture, horticulture, biomass for theproduction of biofuel molecules and other chemicals, and/or forestry.Some examples of these plants may include pineapple, banana, coconut,lily, grasspeas and grass; and dicotyledonous plants, such as, forexample, peas, alfalfa, tomatillo, melon, chickpea, chicory, clover,kale, lentil, soybean, tobacco, potato, sweet potato, radish, cabbage,rape, apple trees, grape, cotton, sunflower, thale cress, canola, citrus(including orange, mandarin, kumquat, lemon, lime, grapefruit,tangerine, tangelo, citron, and pomelo), pepper, bean, lettuce, Panicumvirgatum (switch), Sorghum bicolor (sorghum, sudan), Miscanthusgiganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera(poplar), Zea mays (corn), Glycine max (soybean), Brassica napus(canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryzasativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa),Beta vulgaris (sugarbeet), Pennisetum glaucum (pearl millet), Panicumspp. Sorghum spp., Miscanthus spp., Saccharum spp., Erianthus spp.,Populus spp., Secale cereale (rye), Salix spp. (willow), Eucalyptus spp.(eucalyptus), Triticosecale spp. (triticum-25 wheat X rye), Bamboo,Carthamus tinctorius (safflower), Jatropha curcas (Jatropha), Ricinuscommunis (castor), Elaeis guineensis (oil palm), Phoenix dactylifera(date palm), Archontophoenix cunninghamiana (king palm), Syagrusromanzoffiana (queen palm), Linum usitatissimum (flax), Brassica juncea,Manihot esculenta (cassaya), Lycopersicon esculentum (tomato), Lactucasaliva (lettuce), Musa paradisiaca (banana), Solanum tuberosum (potato),Brassica oleracea (broccoli, cauliflower, brussel sprouts), Camelliasinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa),Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus(pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion),Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima(squash), Cucurbita moschata (squash), Spinacea oleracea (spinach),Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), Solanummelongena (eggplant), Papaver somniferum (opium poppy), Papaverorientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabissaliva, Camptotheca acuminate, Catharanthus roseus, Vinca rosea,Cinchona officinalis, Coichicum autumnale, Veratrum californica,Digitalis lanata, Digitalis purpurea, Dioscorea 5 spp., Andrographispaniculata, Atropa belladonna, Datura stomonium, Berberis spp.,Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca,Galanthus wornorii, Scopolia spp., Lycopodium serratum (Huperziaserrata), Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp.,Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis,Chrysanthemum parthenium, Coleus forskohlii, Tanacetum parthenium,Parthenium argentatum (guayule), Hevea spp. (rubber), Mentha spicata(mint), Mentha piperita (mint), Bixa orellana, Alstroemeria spp., Rosaspp. (rose), Dianthus caryophyllus (carnation), Petunia spp. (petunia),Poinsettia pulcherrima (poinsettia), Nicotiana tabacum (tobacco),Lupinus albus (lupin), Uniola paniculata (oats), Hordeum vulgare(barley), and Lolium spp. (rye).

In some examples, a monocotyledonous plant may be used. Monocotyledonousplants belong to the orders of the Alismatales, Arales, Arecales,Bromeliales, Commelinales, Cyclanthales, Cyperales, Eriocaulales,Hydrocharitales, Juncales, Lilliales, Najadales, Orchidales, Pandanales,Poales, Restionales, Triuridales, Typhales, and Zingiberales. Plantsbelonging to the class of the Gymnospermae are Cycadales, Ginkgoales,Gnetales, and Pinales. In some examples, the monocotyledonous plant canbe selected from the group consisting of a maize, rice, wheat, barley,and sugarcane.

In some examples, a dicotyledonous plant may be used, including thosebelonging to the orders of the Aristochiales, Asterales, Batales,Campanulales, Capparales, Caryophyllales, Casuarinales, Celastrales,Comales, Diapensales, Dilleniales, Dipsacales, Ebenales, Ericales,Eucomiales, Euphorbiales, Fabales, Fagales, Gentianales, Geraniales,Haloragales, Hamamelidales, Middles, Juglandales, Lamiales, Laurales,Lecythidales, Leitneriales, Magniolales, Malvales, Myricales, Myrtales,Nymphaeales, Papeverales, Piperales, Plantaginales, Plumb aginales,Podostemales, Polemoniales, Polygalales, Polygonales, Primulales,Proteales, Rafflesiales, Ranunculales, Rhamnales, Rosales, Rubiales,Salicales, Santales, Sapindales, Sarraceniaceae, Scrophulariales,Theales, Trochodendrales, Umbellales, Urticales, and Violates. In someexamples, the dicotyledonous plant can be selected from the groupconsisting of cotton, soybean, pepper, and tomato.

In some cases, the plant to be improved is not readily amenable toexperimental conditions. For example, a crop plant may take too long togrow enough to practically assess an improved trait serially overmultiple iterations. Accordingly, a first plant from which bacteria areinitially isolated, and/or the plurality of plants to which geneticallymanipulated bacteria are applied may be a model plant, such as a plantmore amenable to evaluation under desired conditions. Non-limitingexamples of model plants include Setaria, Brachypodium, and Arabidopsis.Ability of bacteria isolated according to a method of the disclosureusing a model plant may then be applied to a plant of another type (e.g.a crop plant) to confirm conferral of the improved trait.

Traits that may be improved by the methods disclosed herein include anyobservable characteristic of the plant, including, for example, growthrate, height, weight, color, taste, smell, changes in the production ofone or more compounds by the plant (including for example, metabolites,proteins, drugs, carbohydrates, oils, and any other compounds).Selecting plants based on genotypic information is also envisaged (forexample, including the pattern of plant gene expression in response tothe bacteria, or identifying the presence of genetic markers, such asthose associated with increased nitrogen fixation). Plants may also beselected based on the absence, suppression or inhibition of a certainfeature or trait (such as an undesirable feature or trait) as opposed tothe presence of a certain feature or trait (such as a desirable featureor trait).

Non-Genetically Modified Maize

The methods and bacteria described herein are suitable for any of avariety of non-genetically modified maize plants or part thereof. And insome aspects the corn is organic. Furthermore, the methods and bacteriadescribed herein are suitable for any of the following non-geneticallymodified hybrids, varieties, lineages, etc. In some embodiments, cornvarieties generally fall under six categories: sweet corn, flint corn,popcorn, dent corn, pod corn, and flour corn.

Sweet Corn

Yellow su varieties include Earlivee, Early Sunglow, Sundance, EarlyGolden Bantam, Iochief, Merit, Jubilee, and Golden Cross Bantam. Whitesu varieties include True Platinum, Country Gentleman, Silver Queen, andStowell's Evergreen. Bicolor su varieties include Sugar & Gold, Quickie,Double Standard, Butter & Sugar, Sugar Dots, Honey & Cream. Multicolorsu varieties include Hookers, Triple Play, Painted Hill, BlackMexican/Aztec.

Yellow se varieties include Buttergold, Precocious, Spring Treat, SugarBuns, Colorow, Kandy King, Bodacious R/M, Tuxedo, Incredible, Merlin,Miracle, and Kandy Korn EH. White se varieties include Spring Snow,Sugar Pearl, Whiteout, Cloud Nine, Alpine, Silver King, and Argent.Bicolor se varieties include Sugar Baby, Fleet, Bon Jour, Trinity,Bi-Licious, Temptation, Luscious, Ambrosia, Accord, Brocade, Lancelot,Precious Gem, Peaches and Cream Mid EH, and Delectable R/M. Multicolorse varieties include Ruby Queen.

Yellow sh2 varieties include Extra Early Super Sweet, Takeoff, EarlyXtra Sweet, Raveline, Summer Sweet Yellow, Krispy King, Garrison, IlliniGold, Challenger, Passion, Excel, Jubilee SuperSweet, Illini Xtra Sweet,and Crisp 'N Sweet. White sh2 varieties include Summer Sweet White,Tahoe, Aspen, Treasure, How Sweet It Is, and Camelot. Bicolor sh2varieties include Summer Sweet Bicolor, Radiance, Honey 'N Pearl, Aloha,Dazzle, Hudson, and Phenomenal.

Yellow sy varieties include Applause, Inferno, Honeytreat, and HoneySelect. White sy varieties include Silver Duchess, Cinderella,Mattapoisett, Avalon, and Captivate. Bicolor sy varieties include PayDirt, Revelation, Renaissance, Charisma, Synergy, Montauk, Kristine,Serendipity/Providence, and Cameo.

Yellow augmented supersweet varieties include Xtra-Tender 1ddA,Xtra-Tender 11dd, Mirai 131Y, Mirai 130Y, Vision, and Mirai 002. Whiteaugmented supersweet varieties include Xtra-Tender 3dda, Xtra-Tender31dd, Mirai 421W, XTH 3673, and Devotion. Bicolor augmented supersweetvarieties include Xtra-Tender 2dda, Xtra-Tender 21dd, Kickoff XR, Mirai308BC, Anthem XR, Mirai 336BC, Fantastic XR, Triumph, Mirth 301BC,Stellar, American Dream, Mirai 350BC, and Obsession.

Flint Corn

Flint corn varieties include Bronze-Orange, Candy Red Flint, FlorianiRed Flint, Glass Gem, Indian Ornamental (Rainbow), Mandan Red Flour,Painted Mountain, Petmecky, Cherokee White Flour,

PopCorn

Pop corn varieties include Monarch Butterfly, Yellow Butterfly, MidnightBlue, Ruby Red, Mixed Baby Rice, Queen Mauve, Mushroom Flake, JapaneseHull-less, Strawberry, Blue Shaman, Miniature Colored, Miniature Pink,Pennsylvania Dutch Butter Flavor, and Red Strawberry.

Dent Corn

Dent corn varieties include Bloody Butcher, Blue Clarage, Ohio BlueClarage, Cherokee White Eagle, Hickory Cane, Hickory King, JellicorseTwin, Kentucky Rainbow, Daymon Morgan's Knt. Butcher, Leaming, Leaming'sYellow, McCormack's Blue Giant, Neal Paymaster, Pungo Creek Butcher,Reid's Yellow Dent, Rotten Clarage, and Tennessee Red Cob.

In some embodiments, corn varieties include P1618W, P1306W, P1345,P1151, P1197, P0574, P0589, and P0157. W=white corn.

In some embodiments, the methods and bacteria described herein aresuitable for any hybrid of the maize varieties setforth herein.

Genetically Modified Maize

The methods and bacteria described herein are suitable for any of ahybrid, variety, lineage, etc. of genetically modified maize plants orpart thereof.

Furthermore, the methods and bacteria described herein are suitable forany of the following genetically modified maize events, which have beenapproved in one or more countries: 32138 (32138 SPT Maintainer), 3272(ENOGEN), 3272 x Bt11, 3272 x bt11 x GA21, 3272 x Bt11 x MIR604, 3272 xBt11 x MIR604 x GA21, 3272 x Bt11 x MIR604 x TC1507 x 5307 x GA21, 3272x GA21, 3272 x MIR604, 3272 x MIR604 x GA21, 4114, 5307 (AGRISUREDuracade), 5307 x GA21, 5307 x MIR604 x Bt11 x TC1507 x GA21 (AGRISUREDuracade 5122), 5307 x MIR604 x Bt11 x TC1507 x GA21 x MIR162 (AGRISUREDuracade 5222), 59122 (HERCULEX RW), 59122 x DAS40278, 59122 x GA21,59122 x MIR604, 59122 x MIR604 x GA21, 59122 x MIR604 x TC1507, 59122 xMIR604 x TC1507 x GA21, 59122 x MON810, 59122 x MON810 x MIR604, 59122 xMON810 x NK603, 59122 x MON810 x NK603 x MIR604, 59122 x MON88017, 59122x MON88017 x DAS40278, 59122 x NK603 (Herculex RW ROUNDUP READY 2),59122 x NK603 x MIR604, 59122 x TC1507 x GA21, 676, 678, 680, 3751 IR,98140, 98140 x 59122, 98140 x TC1507, 98140 x TC1507 x 59122, Bt10(Bt10), Bt11 [X4334CBR, X4734CBR] (AGRISURE CB/LL), Bt11 x 5307, Bt11 x5307 x GA21, Bt11 x 59122 x MIR604, Br11 x 59122 x MIR604 x GA21, Bt11 x59122 x MIR604 x TC1507, M53, M56, DAS-59122-7, Bt11 x 59122 x MIR604 xTC1507 x GA21, Bt11 x 59122 x TC1507, TC1507 x DAS-59122-7, Bt11 x 59122x TC1507 x GA21, Bt11 x GA21 (AGRISURE GT/CB/LL), Bt11 x MIR162(AGRISURE Viptera 2100), BT11 x MIR162 x 5307, Bt11 x MIR162 x 5307 xGA21, Bt11 x MIR162 x GA21 (AGRISURE Viptera 3110), Bt11 x MIR162 xMIR604 (AGRISURE Viptera 3100), Bt11 x MIR162 x MIR604 x 5307, Bt11 xMIR162 x MIR604 x 5307 x GA21, Bt11 x MIR162 x MIR604 x GA21 (AGRISUREViptera 3111/AGRISURE Viptera 4), Bt11, MIR162 x MIR604 x MON89034 x5307 x GA21, Bt11 x MIR162 x MIR604 x TC1507, Bt11 x MIR162 x MIR604 xTC1507 x 5307, Bt11 x MIR162 x MIR604 x TC1507 x GA21, Bt11 x MIR162 xMON89034, Bt11 x MIR162 x MON89034 x GA21, Bt11 x MIR162 x TC1507, Bt11x MIR162 x TC1507 x 5307, Bt11 x MIR162 x TC1507 x 5307 x GA21, Bt11 xMR162 x TC1507 x GA21 (AGRISURE Viptera 3220), BT11 x MIR604 (AgrisureBC/LL/RW), Bt11 x MIR604 x 5307, Bt11 x MIR604 x 5307 x GA21, Bt11 xMIR604 x GA21, Bt11 x MIR604 x TC1507, Bt11 x MIR604 x TC1507 x 5307,Bt11 x MIR604 x TC1507 x GA21, Bt11 x MON89034 x GA21, Bt11 x TC1507,Bt11 x TC1507 x 5307, Bt11 x TC1507 x GA21, Bt176 (NaturGardKnockOut/Maximizer), BVLA430101, CBH-351 (STARLINK Maize), DAS40278(ENLIST Maize), DAS40278 x NK603, DBT418 (Bt Xtra Maize), DLL25 [B16],GA21 (ROUNDUP READY Maize/AGRISURE GT), GA21 x MON810 (ROUNDUP READYYieldgard Maize), GA21 x T25, HCEM485, LY038 (MAVERA Maize), LY038 xMON810 (MAVERA Yieldgard Maize), MIR162 (AGRISURE Viptera), MIR162 x5307, MIR162 x 5307 x GA21, MIR162 x GA21, MIR162 x MIR604, MIR162 xMIR604 x 5307, MIR162 x MIR604 x 5307 x GA21, MIR162 x MIR604 x GA21,MIR162 x MIR604 x TC1507 x 5307, MIR162 x MIR604 x TC1507 x 5307 x GA21,MIR162 x MIR604 x TC1507 x GA21, MIR162 x MON89Ø34, MIR162 x NK603,MIR162 x TC1507, MIR162 x TC1507 x 5307, MIR162 x TC1507 x 5307 x GA21,MIR162 x TC1507 x GA21, MIR604 (AGRISURE RW), MIR604 x 5307, MIR604 x5307 x GA21, MIR604 x GA21 (AGRISURE GT/RW), MIR604 x NK603, MIR604 xTC1507, MIR604 x TC1507 x 5307, MIR604 x TC1507 x 5307 x GA21, MIR604 xTC1507 x GA21, MON801 [MON80100], MON802, MON809, MON810 (YIELDGARD,MAIZEGARD), MON810 x MIR162, MON810 x MIR162 x NK603, MON810 x MIR604,MON810 x MON88017 (YIELDGARD VT Triple), MON810 x NK603 x MIR604, MON832(ROUNDUP READY Maize), MON863 (YIELDGARD Rootworm RW, MAXGARD), MON863 xMON810 (YIELDGARD Plus), MON863 x MON810 x NK603 (YIELDGARD Plus withRR), MON863 x NK603 (YIELDGARD RW+RR), MON87403, MON87411, MON87419,MON87427 (ROUNDUP READY Maize), MON87427 x 59122, MON87427 x MON88017,MON87427 x MON88017 x 59122, MON87427 x MON89034, MON87427 x MON89034 x59122, MON87427 x MON89034 x MIR162 x MON87411, MON87427 x MON89034 xMON88017, MON87427 x MON89034 x MON88017 x 59122, MON87427 x MON89034 xNK603, MON87427 x MON89034 x TC1507, MON87427 x MON89034 x TC1507 x59122, MON87427 x MON89034 x TC1507 x MON87411 x 59122, MON87427 xMON89034 x TC1507 x MON87411 x 59122 x DAS40278, MON87427 x MON89034 xTC1507 x MON88017, MON87427 x MON89034 x MIR162 x NK603, MON87427 xMON89034 x TC15Ø7 x MON88017 x 59122, MON87427 x TC1507, MON87427 xTC1507 x 59122, MON87427 x TC1507 x MON88017, MON87427 x TC1507 xMON88017 x 59122, MON87460 (GENUITY DROUGHTGARD), MON87460 x MON88017,MON87460 x MON89034 x MON88017, MON87460 x MON89034 x NK603, MON87460 xNK603, MON88017, MON88017 x DAS40278, MON89034, MON89034 x 59122,MON89034 x 59122 x DAS40278, MON89034 x 59122 x MON88017, MON89034 x59122 x MON88017 x DAS40278, MON89034 x DAS40278, MON89034 x MON87460,MON89034 x MON88017 (GENUITY VT Triple Pro), MON89034 x MON88017 xDAS40278, MON89034 x NK603 (GENUITY VT Double Pro), MON89034 x NK603 xDAS40278, MON89034 x TC1507, MON89034 x TC1507 x 59122, MON89034 xTC1507 x 59122 x DAS40278, MON89034 x TC1507 x DAS40278, MON89034 xTC1507 x MON88017, MON89034 x TC1507 x MON88017 x 59122 (GENUITYSMARTSTAX), MON89034 x TC1507 x MON88017 x 59122 x DAS40278, MON89034 xTC1507 x MON88017 x DAS40278, MON89034 x TC1507 x NK603 (POWER CORE),MON89034 x TC1507 x NK603 x DAS40278, MON89034 x TC1507 x NK603 xMIR162, MON89034 x TC1507 x NK603 x MIR162 x DAS40278, MON89034 x GA21,MS3 (INVIGOR Maize), MS6 (INVIGOR Maize), MZHG0JG, MZIR098, NK603(ROUNDUP READY 2 Maize), NK603 x MON810 x 4114 x MIR604, NK603 x MON810(YIELDGARD CB+RR), NK603 x T25 (ROUNDUP READY LIBERTY LINK Maize), T14(LIBERTY LINK Maize), T25 (LIBERTY LINK Maize), T25 x MON810 (LIBERTYLINK YIELDGARD Maize), TC1507 (HERCULEX I, HERCULEX CB), TC1507 x 59122x MON810 x MIR604 x NK603 (OPTIMUM INTRASECT XTREME), TC1507 x MON810 xMIR604 x NK603, TC1507 x 5307, TC1507 x 5307 x GA21, TC1507 x 59122(HERCULEX XTRA), TC1507 x 59122 x DAS40278, TC1507 x 59122 x MON810,TC1507 x 59122 x MON810 x MIR604, TC1507 x 59122 x MON810 x NK603(OPTIMUM INTRASECT XTRA), TC1507 x 59122 x MON88017, TC1507 x 59122 xMON88017 x DAS40278, TC1507 x 59122 x NK603 (HERCULEX XTRA RR), TC1507 x59122 x NK603 x MIR604, TC1507 x DAS40278, TC1507 x GA21, TC1507 xMIR162 x NK603, TC1507 x MIR604 x NK603 (OPTIMUM TRISECT), TC1507 xMON810, TC1507 x MON810 x MIR162, TC1507 x MON810 x MIR162 x NK603,TC1507 x MON810 x MIR604, TC1507 x MON810 x NK603 (OPTIMUM INTRASECT),TC1507 x MON810 x NK603 x MIR604, TC1507 x MON88017, TC1507 x MON88017 xDAS40278, TC1507 x NK603 (HERCULEX I RR), TC1507 x NK603 x DAS40278,TC6275, and VCO-01981-5.

Additional Genetically Modified Plants

The methods and bacteria described herein are suitable for any of avariety of genetically modified plants or part thereof.

Furthermore, the methods and bacteria described herein are suitable forany of the following genetically modified plant events which have beenapproved in one or more countries.

TABLE 14 Rice Traits, which can be combined with microbes of thedisclosure Oryza sativa Rice Event Company Description CL121, CL141,CFX51 BASF Inc. Tolerance to the imidazolinone herbicide, imazethapyr,induced by chemical mutagenesis of the acetolactate synthase (ALS)enzyme using ethyl methanesulfonate (EMS). IMINTA-1, IMINTA-4 BASF Inc.Tolerance to imidazolinone herbicides induced by chemical mutagenesis ofthe acetolactate synthase (ALS) enzyme using sodium azide. LLRICE06,LLRICE62 Aventis CropScience Glufosinate ammonium herbicide tolerantrice produced by inserting a modified phosphinothricin acetyltransferase(PAT) encoding gene from the soil bacterium Streptomyces hygroscopicus).LLRICE601 Bayer CropScience (Aventis Glufosinate ammonium herbicideCropScience(AgrEvo)) tolerant rice produced by inserting a modifiedphosphinothricin acetyltransferase (PAT) encoding gene from the soilbacterium Streptomyces hygroscopicus). PWC16 BASF Inc. Tolerance to theimidazolinone herbicide, imazethapyr, induced by chemical mutagenesis ofthe acetolactate synthase (ALS) enzyme using ethyl methanesulfonate(EMS).

TABLE 15 Alfalfa Traits, which can be combined with microbes of thedisclosure Medicago sativa Alfalfa Event Company Description J101, J163Monsanto Company and Glyphosate herbicide tolerant Forage Geneticsalfalfa (lucerne) produced by International inserting a gene encodingthe enzyme 5-enolypyruvylshikimate- 3-phosphate synthase (EPSPS) fromthe CP4 strain of Agrobacterium tumefaciens.

TABLE 16 Wheat Traits, which can be combined with microbes of thedisclosure Triticum aestivum Wheat Event Company Description AP205CLBASF Inc. Selection for a mutagenized version of the enzymeacetohydroxyacid synthase (AHAS), also known as acetolactate synthase(ALS) or acetolactate pyruvate-lyase. AP602CL BASF Inc. Selection for amutagenized version of the enzyme acetohydroxyacid synthase (AHAS), alsoknown as acetolactate synthase (ALS) or acetolactate pyruvate-lyase.BW255-2, BW238-3 BASF Inc. Selection for a mutagenized version of theenzyme acetohydroxyacid synthase (AHAS), also known as acetolactatesynthase (ALS) or acetolactate pyruvate-lyase. BW7 BASF Inc. Toleranceto imidazolinone herbicides induced by chemical mutagenesis of theacetohydroxyacid synthase (AHAS) gene using sodium azide. MON71800Monsanto Company Glyphosate tolerant wheat variety produced by insertinga modified 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS) encodinggene from the soil bacterium Agrobacterium tumefaciens, strain CP4.SWP965001 Cyanamid Crop Selection for a mutagenized version Protectionof the enzyme acetohydroxyacid synthase (AHAS), also known asacetolactate synthase (ALS) or acetolactate pyruvate-lyase. Teal 11ABASF Inc. Selection for a mutagenized version of the enzymeacetohydroxyacid synthase (AHAS), also known as acetolactate synthase(ALS) or acetolactate pyruvate-lyase.

TABLE 17 Sunflower Traits, which can be combined with microbes of thedisclosure Helianthus annuus Sunflower Event Company Description X81359BASF Inc. Tolerance to imidazolinone herbicides by selection of anaturally occurring mutant.

TABLE 18 Soybean Traits, which can be combined with microbes of thedisclosure Glycine max L. Soybean Event Company Description A2704-12,A2704-21, Bayer CropScience Glufosinate ammonium herbicide A5547-35(Aventis CropScience tolerant soybean produced by (AgrEvo)) inserting amodified phosphinothricin acetyltransferase (PAT) encoding gene from thesoil bacterium Streptomyces viridochromogenes. A5547-127 BayerCropScience Glufosinate ammonium herbicide (Aventis CropScience tolerantsoybean produced by (AgrEvo)) inserting a modified phosphinothricinacetyltransferase (PAT) encoding gene from the soil bacteriumStreptomyces viridochromogenes. BPS-CV127-9 BASF Inc. The introducedcsr1-2 gene from Arabidopsis thaliana encodes an acetohydroxyacidsynthase protein that confers tolerance to imidazolinone herbicides dueto a point mutation that results in a single amino acid substitution inwhich the serine residue at position 653 is replaced by asparagine(S653N). DP-305423 Pioneer Hi-Bred High oleic acid soybean producedInternational Inc. by inserting additional copies of a portion of theomega 6 desaturase encoding gene, gm-fad2-1 resulting in silencing ofthe endogenous omega-6 desaturase gene (FAD2-1). DP356043 PioneerHi-Bred Soybean event with two herbicide International Inc. tolerancegenes: glyphosate N- acetlytransferase, which detoxifies glyphosate, anda modified acetolactate synthase (ALS) gene which is tolerant toALS-inhibiting herbicides. G94-1, G94-19, G168 DuPont Canada High oleicacid soybean produced Agricultural Products by inserting a second copyof the fatty acid desaturase (Gm Fad2-1) encoding gene from soybean,which resulted in “silencing” of the endogenous host gene. GTS 40-3-2Monsanto Company Glyphosate tolerant soybean variety produced byinserting a modified 5- enolpyruvylshikimate-3-phosphate synthase(EPSPS) encoding gene from the soil bacterium Agrobacterium tumefaciens.GU262 Bayer CropScience Glufosinate ammonium herbicide (Aventis tolerantsoybean produced by CropScience(AgrEvo)) inserting a modifiedphosphinothricin acetyltransferase (PAT) encoding gene from the soilbacterium Streptomyces viridochromogenes. MON87701 Monsanto CompanyResistance to Lepidopteran pests of soybean including velvetbeancaterpillar (Anticarsia gemmatalis) and soybean looper (Pseudoplusiaincludens). MON87701 × Monsanto Company Glyphosate herbicide toleranceMON89788 through expression of the EPSPS encoding gene from A.tumefaciens strain CP4, and resistance to Lepidopteran pests of soybeanincluding velvetbean caterpillar (Anticarsia gemmatalis) and soybeanlooper (Pseudoplusia includens) via expression of the Cry1Ac encodinggene from B. thuringiensis. MON89788 Monsanto CompanyGlyphosate-tolerant soybean produced by inserting a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding aroA (epsps)gene from Agrobacterium tumefaciens CP4. OT96-15 Agriculture & Agri-FoodLow linolenic acid soybean Canada produced through traditional cross-breeding to incorporate the novel trait from a naturally occurring fan1gene mutant that was selected for low linolenic acid. W62, W98 BayerCropScience Glufosinate ammonium herbicide (Aventis tolerant soybeanproduced by CropScience(AgrEvo)) inserting a modified phosphinothricinacetyltransferase (PAT) encoding gene from the soil bacteriumStreptomyces hygroscopicus.

TABLE 19 Corn Traits, which can be combined with microbes of thedisclosure Zea mays L. Maize Event Company Description 176 SyngentaSeeds, Inc. Insect-resistant maize produced by inserting the Cry1Ab genefrom Bacillus thuringiensis subsp. kurstaki. The genetic modificationaffords resistance to attack by the European corn borer (ECB). 3751 IRPioneer Hi-Bred Selection of somaclonal variants 676, 678, 680International Inc. by culture of embryos on Pioneer Hi-Bredimidazolinone containing media. International Inc. Male-sterile andglufosinate ammonium herbicide tolerant maize produced by insertinggenes encoding DNA adenine methylase and phosphinothricinacetyltransferase (PAT) from Escherichia coli and Streptomycesviridochromogenes, respectively. B16 (DLL25) Dekalb Genetics Glufosinateammonium herbicide Corporation tolerant maize produced by inserting thegene encoding phosphinothricin acetyltransferase (PAT) from Streptomyceshygroscopicus. BT11 (X4334CBR, Syngenta Seeds, Inc. Insect-resistant andherbicide X4734CBR) tolerant maize produced by inserting the Cry1Ab genefrom Bacillus thuringiensis subsp. kurstaki, and the phosphinothricinN-acetyltransferase (PAT) encoding gene from S. viridochromogenes. BT11× GA21 Syngenta Seeds, Inc. Stacked insect resistant and herbicidetolerant maize produced by conventional cross breeding of parental linesBT11 (OECD unique identifier: SYN-BTO11-1) and GA21 (OECD uniqueidentifier: MON-OOO21-9). BT11 × MIR162 × Syngenta Seeds, Inc.Resistance to Coleopteran pests, MIR604 × GA21 particularly cornrootworm pests (Diabrotica spp.) and several Lepidopteran pests of corn,including European corn borer (ECB, Ostrinia nubilalis), corn earworm(CEW, Helicoverpa zea), fall army worm (FAW, Spodoptera frugiperda), andblack cutworm (BCW, Agrotis ipsilon); tolerance to glyphosate andglufosinate- ammonium containing herbicides. BT11 × MIR162 SyngentaSeeds, Inc. Stacked insect resistant and herbicide tolerant maizeproduced by conventional cross breeding of parental lines BT11 (OECDunique identifier: SYN-BTO11-1) and MIR162 (OECD unique identifier:SYN-1R162-4). Resistance to the European Corn Borer and tolerance to theherbicide glufosinate ammonium (Liberty) is derived from BT11, whichcontains the Cry1Ab gene from Bacillus thuringiensis subsp. kurstaki,and the phosphinothricin N-acetyltransferase (PAT) encoding gene from S.viridochromogenes. Resistance to other Lepidopteran pests, including H.zea, S. frugiperda, A. ipsilon, and S. albicosta, is derived fromMIR162, which contains the vip3Aa gene from Bacillus thuringiensisstrain AB88. BT11 × MIR162 × Syngenta Seeds, Inc. Bacillus thuringiensisCry1Ab MIR604 delta-endotoxin protein and the genetic material necessaryfor its production (via elements of vector pZO1502) in Event Bt11 corn(OECD Unique Identifier: SYNBTO11-1) × Bacillus thuringiensis Vip3Aa20insecticidal protein and the genetic material necessary for itsproduction (via elements of vector pNOV1300) in Event MIR162 maize (OECDUnique Identifier: SYN-IR162-4) × modified Cry3A protein and the geneticmaterial necessary for its production (via elements of vector pZM26) inEvent MIR604 corn (OECD Unique Identifier: SYN-1R604-5). CBH-351 AventisCropScience Insect-resistant and glufosinate ammonium herbicide tolerantmaize developed by inserting genes encoding Cry9C protein from Bacillusthuringiensis subsp tolworthi and phosphinothricin acetyltransferase(PAT) from Streptomyces hygroscopicus. DAS-06275-8 DOW AgroSciences LLCLepidopteran insect resistant and glufosinate ammonium herbicide-tolerant maize variety produced by inserting the Cry1F gene fromBacillus thuringiensis var aizawai and the phosphinothricinacetyltransferase (PAT) from Streptomyces hygroscopicus. BT11 × MIR604Syngenta Seeds, Inc. Stacked insect resistant and herbicide tolerantmaize produced by conventional cross breeding of parental lines BT11(OECD unique identifier: SYN-BTO11-1) and MIR604 (OECD uniqueidentifier: SYN-1R6O5-5). Resistance to the European Corn Borer andtolerance to the herbicide glufosinate ammonium (Liberty) is derivedfrom BT11, which contains the Cry1Ab gene from Bacillus thuringiensissubsp. kurstaki, and the phosphinothricin N-acetyltransferase (PAT)encoding gene from S. viridochromogenes. Corn rootworm -resistance isderived from MIR604 which contains the mCry3A gene from Bacillusthuringiensis. BT11 × MIR604 × Syngenta Seeds, Inc. Stacked insectresistant and GA21 herbicide tolerant maize produced by conventionalcross breeding of parental lines BT11 (OECD unique identifier:SYN-BTO11-1), MIR604 (OECD unique identifier: SYN-1R6O5-5) and GA21(OECD unique identifier: MON- OOO21-9). Resistance to the European CornBorer and tolerance to the herbicide glufosinate ammonium (Liberty) isderived from BT11, which contains the Cry1Ab gene from Bacillusthuringiensis subsp. kurstaki, and the phosphinothricinN-acetyltransferase (PAT) encoding gene from S. viridochromogenes. Cornrootworm-resistance is derived from MIR604 which contains the mCry3Agene from Bacillus thuringiensis. Tolerance to glyphosate herbicide isderived from GA21 which contains a a modified EPSPS gene from maize.DAS-59122-7 DOW AgroSciences LLC Corn rootworm-resistant maize andPioneer Hi-Bred produced by inserting the International Inc. Cry34Ab1and Cry35Ab1 genes from Bacillus thuringiensis strain PS149B1. The PATencoding gene from Streptomyces viridochromogenes was introduced as aselectable marker. DAS-59122-7 × DOW AgroSciences LLC Stacked insectresistant and TC1507 × NK603 and Pioneer Hi-Bred herbicide tolerantmaize produced International Inc. by conventional cross breeding ofparental lines DAS-59122-7 (OECD unique identifier: DAS- 59122-7) andTC1507 (OECD unique identifier: DAS-01507-1) with NK603 (OECD uniqueidentifier: MON-00603-6). Corn rootworm-resistance is derived fromDAS-59122- 7 which contains the Cry34Abl and Cry35Abl genes fromBacillus thuringiensis strain P5149B1. Lepidopteran resistance andtolerance to glufosinate ammonium herbicide is derived from TC1507.Tolerance to glyphosate herbicide is derived from NK603. DBT418 DekalbGenetics Insect-resistant and glufosinate Corporation ammonium herbicidetolerant maize developed by inserting genes encoding Cry1AC protein fromBacillus thuringiensis subsp kurstaki and phosphinothricinacetyltransferase (PAT) from Streptomyces hygroscopicus. MIR604 × GA21Syngenta Seeds, Inc. Stacked insect resistant and herbicide tolerantmaize produced by conventional cross breeding of parental lines MIR604(OECD unique identifier: SYN-1R605-5) and GA21 (OECD unique identifier:MON-00021-9). Corn rootworm-resistance is derived from MIR604 whichcontains the mCry3A gene from Bacillus thuringiensis. Tolerance toglyphosate herbicide is derived from GA21. MON80100 Monsanto CompanyInsect-resistant maize produced by inserting the Cry1Ab gene fromBacillus thuringiensis subsp. kurstaki. The genetic modification affordsresistance to attack by the European corn borer (ECB). MON802 MonsantoCompany Insect-resistant and glyphosate herbicide tolerant maizeproduced by inserting the genes encoding the Cry1Ab protein fromBacillus thuringiensis and the 5- enolpyruvylshikimate-3-phosphatesynthase (EPSPS) from A. tumefaciens strain CP4. MON809 Pioneer Hi-BredResistance to European corn borer International Inc. (Ostrinianubilalis) by introduction of a synthetic Cry1Ab gene. Glyphosateresistance via introduction of the bacterial version of a plant enzyme,5-enolpynivyl shikimate-3- phosphate synthase (EPSPS). MON810 MonsantoCompany Insect-resistant maize produced by inserting a truncated form ofthe Cry1Ab gene from Bacillus thuringiensis subsp. kurstaki HD-1. Thegenetic modification affords resistance to attack by the European cornborer (ECB). MON810 × LY038 Monsanto Company Stacked insect resistantand enhanced lysine content maize derived from conventionalcrossbreeding of the parental lines MON810 (OECD identifier:MON-OO81O-6) and LY038 (OECD identifier: REN-OOO38-3). MON810 × MON88017Monsanto Company Stacked insect resistant and glyphosate tolerant maizederived from conventional cross-breeding of the parental lines MON810(OECD identifier: MON-OO81O- 6) and MON88017 (OECD identifier:MON-88017-3). European corn borer (ECB) resistance is derived from atruncated form of the Cry1Ab gene from Bacillus thuringiensis subsp.kurstaki HD-1 present in MON810. Corn rootworm resistance is derivedfrom the Cry3Bbl gene from Bacillus thuringiensis subspecieskumamotoensis strain EG4691 present in MON88017. Glyphosate tolerance isderived from a 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS)encoding gene from Agrobacterium tumefaciens strain CP4 present inMON88017. MON832 Monsanto Company Introduction, by particle bombardment,of glyphosate oxidase (GOX) and a modified 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme involved in theshikimate biochemical pathway for the production of the aromatic aminoacids. MON863 Monsanto Company Corn rootworm resistant maize produced byinserting the Cry3Bbl gene from Bacillus thuringiensis subsp.kumamotoensis. MON863 × MON810 Monsanto Company Stacked insect resistantcorn hybrid derived from conventional cross-breeding of the parentallines MON863 (OECD identifier: MON-00863-5) and MON810 (OECD identifier:MON-00810-6) MON863 × MON810 × Monsanto Company Stacked insect resistantand Monsanto NK603 herbicide tolerant corn hybrid derived fromconventional crossbreeding of the stacked hybrid MON-00863-5 × MON-00810-6 and NK603 (OECD identifier: MON-00603-6). MON863 × NK603Monsanto Company Stacked insect resistant and herbicide tolerant cornhybrid derived from conventional crossbreeding of the parental linesMON863 (OECD identifier: MON-OO863-5) and NK603 (OECD identifier:MON-OO6O3-6). MON87460 Monsanto Company MON 87460 was developed toprovide reduced yield loss under water-limited conditions compared toconventional maize. Efficacy in MON 87460 is derived by expression ofthe inserted Bacillus subtilis cold shock protein B (CspB). MON88017Monsanto Company Corn rootworm-resistant maize produced by inserting theCry3Bbl gene from Bacillus thuringiensis subspecies kumamotoensis strainEG4691. Glyphosate tolerance derived by inserting a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene fromAgrobacterium tumefaciens strain CP4. MON89034 Monsanto Company Maizeevent expressing two different insecticidal proteins from Bacillusthuringiensis providing resistance to number of Lepidopteran pests.MON89034 × Monsanto Company Stacked insect resistant and MON88017glyphosate tolerant maize derived from conventional cross-breeding ofthe parental lines MON89034 (OECD identifier: MON-89O34-3) and MON88017(OECD identifier: MON-88O17-3). Resistance to Lepidopteran insects isderived from two Cry genes present in MON89043. Corn rootworm resistanceis derived from a single Cry genes and glyphosate tolerance is derivedfrom the 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encodinggene from Agrobacterium tumefaciens present in MON88017. MON89034 ×NK603 Monsanto Company Stacked insect resistant and herbicide tolerantmaize produced by conventional cross breeding of parental lines MON89034(OECD identifier: MON-89034-3) with NK603 (OECD unique identifier:MON-00603-6). Resistance to Lepidopteran insects is derived from two Crygenes present in MON89043. Tolerance to glyphosate herbicide is derivedfrom NK603. NK603 × MON810 Monsanto Company Stacked insect resistant andherbicide tolerant corn hybrid derived from conventional crossbreedingof the parental lines NK603 (OECD identifier: MON- 00603-6) and MON810(OECD identifier: MON-00810-6). MON89034 × TC1507 × Monsanto Company andStacked insect resistant and MON88017 × DAS- Mycogen Seeds c/o Dowherbicide tolerant maize produced 59122-7 AgroSciences LLC byconventional cross breeding of parental lines: MON89034, TC1507,MON88017, and DAS-59 122. Resistance to the above- ground andbelow-ground insect pests and tolerance to glyphosate andglufosinate-ammonium containing herbicides. M53 Bayer CropScience Malesterility caused by expression (Aventis of the barnase ribonuclease geneCropScience(AgrEvo)) from Bacillus amyloliquefaciens; PPT resistance wasvia PPT- acetyltransferase (PAT). M56 Bayer CropScience Male sterilitycaused by expression (Aventis of the barnase ribonuclease geneCropScience(AgrEvo) from Bacillus amyloliquefaciens; PPT resistance wasvia PPT- acetyltransferase (PAT). NK603 Monsanto Company Introduction,by particle bombardment, of a modified 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme involved in theshikimate biochemical pathway for the production of the aromatic aminoacids. NK603 × T25 Monsanto Company Stacked glufosinate ammonium andglyphosate herbicide tolerant maize hybrid derived from conventionalcross-breeding of the parental lines NK603 (OECD identifier:MON-00603-6) and T25 (OECD identifier: ACS-ZM003-2). T25 × MON810 BayerCropScience Stacked insect resistant and (Aventis herbicide tolerantcorn hybrid CropScience(AgrEvo)) derived from conventional crossbreedingof the parental lines T25 (OECD identifier: ACS- ZMOO3-2) and MON810(OECD identifier: MON-OO81O-6). TC1507 Mycogen (c/o Dow Insect-resistantand glufosinate AgroSciences); Pioneer ammonium herbicide tolerant (c/oDuPont) maize produced by inserting the Cry1F gene from Bacillusthuringiensis var. aizawai and the phosphinothricin N-acetyltransferaseencoding gene from Streptomyces viridochromogenes. TC1507 × NK603 DOWAgroSciences LLC Stacked insect resistant and herbicide tolerant cornhybrid derived from conventional crossbreeding of the parental lines1507 (OECD identifier: DAS- O15O7-1) and NK603 (OECD identifier:MON-OO6O3-6). TC1507 × DAS-59122-7 DOW AgroSciences LLC Stacked insectresistant and and Pioneer Hi-Bred herbicide tolerant maize producedInternational Inc. by conventional cross breeding of parental linesTC1507 (OECD unique identifier: DAS-O15O7-1) with DAS-59122-7 (OECDunique identifier: DAS-59122-7). Resistance to Lepidopteran insects isderived from TC1507 due the presence of the Cry1F gene from Bacillusthuringiensis var. aizawai. Corn rootworm-resistance is derived fromDAS-59122-7 which contains the Cry34Ab1 and Cry35Ab1 genes from Bacillusthuringiensis strain P5149B1. Tolerance to glufosinate ammoniumherbicide is derived from TC1507 from the phosphinothricinN-acetyltransferase encoding gene from Streptomyces viridochromogenes.Event Company Description Hybrid Family P0157 Dupont Pioneer P0157P0157AM Dupont Pioneer AM LL RR2 P0157 P0157AMXT Dupont Pioneer AMXT LLRR2 P0157 P0157R Dupont Pioneer RR2 P0157 P0339AM Dupont Pioneer AM LLRR2 P0339 P0339AMXT Dupont Pioneer AMXT LL RR2 P0339 P0306AM DupontPioneer AM LL RR2 P0306 P0589 Dupont Pioneer P0589 P0589AM DupontPioneer AM LL RR2 P0589 P0589AMXT Dupont Pioneer AMXT LL RR2 P0589P0589R Dupont Pioneer RR2 P0589 P0574 Dupont Pioneer P0574 P0574AMDupont Pioneer AM LL RR2 P0574 P0574AMXT Dupont Pioneer AMXT LL RR2P0574 P0533EXR Dupont Pioneer HXX LL RR2 P0533 P0506AM Dupont Pioneer AMLL RR2 P0566 P0760AMXT Dupont Pioneer AMXT LL RR2 P0760 P0707AM DupontPioneer AM LL RR2 P0707 P0707AMXT Dupont Pioneer AMXT LL RR2 P0707P0825AM Dupont Pioneer AM LL RR2 P0825 P0825AMXT Dupont Pioneer AMXT LLRR2 P0825 P0969AM Dupont Pioneer AM LL RR2 P0969 P0969AMXT DupontPioneer AMXT LL RR2 P0969 P0937AM Dupont Pioneer AM LL RR2 P0937 P0919AMDupont Pioneer AM LL RR2 P0919 P0905EXR Dupont Pioneer HXX LL RR2 P0905P1197 Dupont Pioneer P1197 P1197AM Dupont Pioneer AM LL RR2 P1197P1197AMXT Dupont Pioneer AMXT LL RR2 P1197 P1197R Dupont Pioneer RR2P1197 P1151 Dupont Pioneer P1151 P1151AM Dupont Pioneer AM LL RR2 P1151P1151R Dupont Pioneer RR2 P1151 P1138AM Dupont Pioneer AM LL RR2 P1138P1366AM Dupont Pioneer AM LL RR2 P1366 P1366AMXT Dupont Pioneer AMXT LLRR2 P1366 P1365AMX Dupont Pioneer AMX LL RR2 P1365 P1353AM DupontPioneer AM LL RR2 P1353 P1345 Dupont Pioneer P1345 P1311AMXT DupontPioneer AMXT LL RR2 P1311 P1498EHR Dupont Pioneer HX1 LL RR2 P1498P1498R Dupont Pioneer RR2 P1498 P1443AM Dupont Pioneer AM LL RR2 P1443P1555CHR Dupont Pioneer RW HX1 LL RR2 P1555 P1751AMT Dupont Pioneer AMTLL RR2 P1751 P2089AM Dupont Pioneer AM LL RR2 P2089 QROME Dupont PioneerQ LL RR2

The following are the definitions for the shorthand occurring in Table19. AM—OPTIMUM ACREMAX Insect Protection system with YGCB, HX1, LL,AMT—OPTIMUM ACREMAX TRISECT insect Protection System with RW, YGCB, HX1,LL, RR2. AMXT—(OPTIMUM ACREMAX XTreme). HXX—HERCULEX XTRA contains theHerculex I and Herculex RW genes. HX—Contains the HERCULEX 1 InsectProtection gene which provides protection against European corn borer,southwestern corn borer, black cutworm, fill armyworm, western beancutworm, lesser corn stalk borer, southern corn stalk borer, andsugarcane borer; and suppresses corn earworm. LL—Contains theLIBERTYLINK gene for resistance to LIBERTY herbicide. RR2—Contains theROUNDUP READY Corn 2 trait that provides crop safety for over-the-topapplications of labeled glyphosate herbicides when applied according tolabel directions. YGCB—contains the YIELDGARD Corn Borer gene offers ahigh level of resistance to European corn borer, southwestern cornborer, and southern cornstalk borer; moderate resistance to corn earwormand common stalk borer; and above average resistance to fall armyworm.RW—contains the AGRISURE rootworm resistance trait. Q—providesprotection or suppression against susceptible European corn borer,southwestern corn borer, black cutworm, fall armyworm, lesser corn stalkborer, southern corn stalk borer, stalk borer, sugarcane borer, and cornearworm; and also provides protection from larval injury caused bysusceptible western corn rootworm, northern corn rootworm, and Mexicancorn rootworm; contains (1) HERCULEX XTRA insect Protection genes thatproduce Cry1F and Cry34ab1 and Cry35ab1 proteins, (2) AGRISURE RW traitthat includes a gene that produces mCry3A protein, and (3) YIELDGARDCorn Borer gene which produces Cry1 Ab protein.

Concentrations and Rates of Application of Agricultural Compositions

As aforementioned, the agricultural compositions of the presentdisclosure, which comprise a taught microbe, can be applied to plants ina multitude of ways. In two particular aspects, the disclosurecontemplates an in-furrow treatment or a seed treatment

For seed treatment embodiments, the microbes of the disclosure can bepresent on the seed in a variety of concentrations. For example, themicrobes can be found in a seed treatment at a cfu concentration, perseed of: 1×10¹, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹,1×10¹⁰, or more. In particular aspects, the seed treatment compositionscomprise about 1×10⁴ to about 1×10⁸ cfu per seed. In other particularaspects, the seed treatment compositions comprise about 1×10⁵ to about1×10⁷ cfu per seed. In other aspects, the seed treatment compositionscomprise about 1×10⁶ cfu per seed.

In the United States, about 10% of corn acreage is planted at a seeddensity of above about 36,000 seeds per acre; ⅓ of the corn acreage isplanted at a seed density of between about 33,000 to 36,000 seeds peracre; ⅓ of the corn acreage is planted at a seed density of betweenabout 30,000 to 33,000 seeds per acre, and the remainder of the acreageis variable. See, “Corn Seeding Rate Considerations,” written by SteveButzen, available at:www.pioneer.com/home/site/us/agronomy/library/corn-seeding-rate-considerations/.

Table 20 below utilizes various cfu concentrations per seed in acontemplated seed treatment embodiment (rows across) and various seedacreage planting densities (1^(st) column: 15K-41K) to calculate thetotal amount of cfu per acre, which would be utilized in variousagricultural scenarios (i.e. seed treatment concentration per seed×seeddensity planted per acre). Thus, if one were to utilize a seed treatmentwith 1×10⁶ cfu per seed and plant 30,000 seeds per acre, then the totalcfu content per acre would be 3×10¹⁰ (i.e. 30K*1×10⁶).

TABLE 20 Total CFU Per Acre Calculation for Seed Treatment EmbodimentsCorn Population (i.e. seeds per acre) 1.00E+02 1.00E+03 1.00E+041.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 15,000 1.50E+06 1.50E+071.50E+08 1.50E+09 1.50E+10 1.50E+11 1.50E+12 1.50E+13 16,000 1.60E+061.60E+07 1.60E+08 1.60E+09 1.60E+10 1.60E+11 1.60E+12 1.60E+13 17,0001.70E+06 1.70E+07 1.70E+08 1.70E+09 1.70E+10 1.70E+11 1.70E+12 1.70E+1318,000 1.80E+06 1.80E+07 1.80E+08 1.80E+09 1.80E+10 1.80E+11 1.80E+121.80E+13 19,000 1.90E+06 1.90E+07 1.90E+08 1.90E+09 1.90E+10 1.90E+111.90E+12 1.90E+13 20,000 2.00E+06 2.00E+07 2.00E+08 2.00E+09 2.00E+102.00E+11 2.00E+12 2.00E+13 21,000 2.10E+06 2.10E+07 2.10E+08 2.10E+092.10E+10 2.10E+11 2.10E+12 2.10E+13 22,000 2.20E+06 2.20E+07 2.20E+082.20E+09 2.20E+10 2.20E+11 2.20E+12 2.20E+13 23,000 2.30E+06 2.30E+072.30E+08 2.30E+09 2.30E+10 2.30E+11 2.30E+12 2.30E+13 24,000 2.40E+062.40E+07 2.40E+08 2.40E+09 2.40E+10 2.40E+11 2.40E+12 2.40E+13 25,0002.50E+06 2.50E+07 2.50E+08 2.50E+09 2.50E+10 2.50E+11 2.50E+12 2.50E+1326,000 2.60E+06 2.60E+07 2.60E+08 2.60E+09 2.60E+10 2.60E+11 2.60E+122.60E+13 27,000 2.70E+06 2.70E+07 2.70E+08 2.70E+09 2.70E+10 2.70E+112.70E+12 2.70E+13 28,000 2.80E+06 2.80E+07 2.80E+08 2.80E+09 2.80E+102.80E+11 2.80E+12 2.80E+13 29,000 2.90E+06 2.90E+07 2.90E+08 2.90E+092.90E+10 2.90E+11 2.90E+12 2.90E+13 30,000 3.00E+06 3.00E+07 3.00E+083.00E+09 3.00E+10 3.00E+11 3.00E+12 3.00E+13 31,000 3.10E+06 3.10E+073.10E+08 3.10E+09 3.10E+10 3.10E+11 3.10E+12 3.10E+13 32,000 3.20E+063.20E+07 3.20E+08 3.20E+09 3.20E+10 3.20E+11 3.20E+12 3.20E+13 33,0003.30E+06 3.30E+07 3.30E+08 3.30E+09 3.30E+10 3.30E+11 3.30E+12 3.30E+1334,000 3.40E+06 3.40E+07 3.40E+08 3.40E+09 3.40E+10 3.40E+11 3.40E+123.40E+13 35,000 3.50E+06 3.50E+07 3.50E+08 3.50E+09 3.50E+10 3.50E+113.50E+12 3.50E+13 36,000 3.60E+06 3.60E+07 3.60E+08 3.60E+09 3.60E+103.60E+11 3.60E+12 3.60E+13 37,000 3.70E+06 3.70E+07 3.70E+08 3.70E+093.70E+10 3.70E+11 3.70E+12 3.70E+13 38,000 3.80E+06 3.80E+07 3.80E+083.80E+09 3.80E+10 3.80E+11 3.80E+12 3.80E+13 39,000 3.90E+06 3.90E+073.90E+08 3.90E+09 3.90E+10 3.90E+11 3.90E+12 3.90E+13 40,000 4.00E+064.00E+07 4.00E+08 4.00E+09 4.00E+10 4.00E+11 4.00E+12 4.00E+13 41,0004.10E+06 4.10E+07 4.10E+08 4.10E+09 4.10E+10 4.10E+11 4.10E+12 4.10E+13

For in-furrow embodiments, the microbes of the disclosure can be appliedat a cfu concentration per acre of: 1×10⁶, 3.20×10¹⁰, 1.60×10¹¹,3.20×10¹¹, 8.0×10¹¹, 1.6×10¹², 3.20×10¹², or more. Therefore, inaspects, the liquid in-furrow compositions can be applied at aconcentration of between about 1×10⁶ to about 3×10¹² cfu per acre.

In some aspects, the in-furrow compositions are contained in a liquidformulation. In the liquid in-furrow embodiments, the microbes can bepresent at a cfu concentration per milliliter of: 1×10¹, 1×10², 1×10³,1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹²,1×10¹³, or more. In certain aspects, the liquid in-furrow compositionscomprise microbes at a concentration of about 1×10⁶ to about 1×10¹¹ cfuper milliliter. In other aspects, the liquid in-furrow compositionscomprise microbes at a concentration of about 1×10⁷ to about 1×10¹⁰ cfuper milliliter. In other aspects, the liquid in-furrow compositionscomprise microbes at a concentration of about 1×10⁸ to about 1×10⁹ cfuper milliliter. In other aspects, the liquid in-furrow compositionscomprise microbes at a concentration of up to about 1×10¹³ cfu permilliliter.

Transcriptomic Profiling of Candidate Microbes

Previous work by the inventors entailed transcriptomic profiling ofstrain CI010 to identify promoters that are active in the presence ofenvironmental nitrogen. Strain CI010 was cultured in a defined,nitrogen-free media supplemented with 10 mM glutamine. Total RNA wasextracted from these cultures (QIAGEN RNeasy kit) and subjected toRNAseq sequencing via Illumina HiSeq (SeqMatic, Fremont Calif.).Sequencing reads were mapped to the CI010 genome data using Geneious,and highly expressed genes under control of proximal transcriptionalpromoters were identified.

Tables 21-23 lists genes and their relative expression level as measuredthrough RNASeq sequencing of total RNA. Sequences of the proximalpromoters were recorded for use in mutagenesis of nif pathways, nitrogenutilization related pathways, or other genes with a desired expressionlevel.

TABLE 21 Name Minimum Maximum Length Direction murein lipoprotein CDS2,929,898 2,930,134 237 forward membrane protein CDS 5,217,517 5,217,843327 forward zinc/cadmium-binding 3,479,979 3,480,626 648 forward proteinCDS acyl carrier protein CDS 4,563,344 4,563,580 237 reverse ompX CDS4,251,002 4,251,514 513 forward DNA-binding protein 375,156 375,428 273forward HU-beta CDS sspA CDS 629,998 630,636 639 reverse taffi CDS3,199,435 3,199,638 204 reverse LexA repressor CDS 1,850,457 1,851,065609 forward hisS CDS <3999979 4,001,223 >1245 forward

TABLE 22 Differential Expression Differential RNASeq_nifL -RNASeq_nifL - RNASeq_WT - RNASeq_WT - Absolute Expression Raw Read RawTranscript Raw Read Raw Transcript Name Confidence Ratio Count CountCount Count murein 1000 −1.8 12950.5 10078.9 5151.5 4106.8 lipoproteinCDS membrane 1000 −1.3 9522.5 5371.3 5400 3120 protein CDS zinc/cadmium-3.3 1.1 6461 1839.1 5318 1550.6 binding protein CDS acyl carrier 25.61.6 1230.5 957.6 1473.5 1174.7 protein CDS ompX CDS 1.7 1.1 2042 734.21687.5 621.5 DNA-binding 6.9 −1.3 1305 881.7 725 501.8 protein HU- betaCDS sspA CDS 0.2 1 654 188.8 504.5 149.2 tatE CDS 1.4 1.3 131 118.4 125115.8 LexA 0.1 −1.1 248 75.1 164 50.9 repressor CDS hisS CDS 0 −1.1 46769.2 325 49.3

TABLE 23 Prm (In Forward direction, −250 Expressed Neighbor to +10region) Sequence Sequence Name SEQ ID NO: SEQ ID NO: SEQ ID NO: mureinlipoprotein CDS SEQ ID NO: 3 SEQ ID NO: 13 SEQ ID NO: 23 membraneprotein CDS SEQ ID NO: 4 SEQ ID NO: 14 SEQ ID NO: 24zinc/cadmium-binding SEQ ID NO: 5 SEQ ID NO: 15 SEQ ID NO: 25 proteinCDS acyl carrier protein CDS SEQ ID NO: 6 SEQ ID NO: 16 SEQ ID NO: 26ompX CDS SEQ ID NO: 7 SEQ ID NO: 17 SEQ ID NO. 27 DNA-binding proteinSEQ ID NO: 8 SEQ ID NO: 18 SEQ ID NO: 28 HU-beta CDS sspA CDS SEQ ID NO:9 SEQ ID NO: 19 SEQ ID NO: 29 tatE CDS SEQ ID NO: 10 SEQ ID NO: 20 SEQID NO: 30 LexA repressor CDS SEQ ID NO: 11 SEQ ID NO: 21 SEQ ID NO: 31hisS CDS SEQ ID NO: 12 SEQ ID NO: 22 SEQ ID NO: 32

TABLE 24 Table of Strains Mutagenic DNA Gene 1 Gene 2 Name LineageDescription Genotype mutation mutation CI006 Isolated strain from NoneWT Enterobacter (now Kosakonia) genera CI008 Isolated strain from NoneWT Burkholderia (now Paraburkholderia) genera CI010 Isolated strain fromNone WT Klebsiella genera CI019 Isolated strain from None WT Rahnellagenera CI028 Isolated strain from None WT Enterobacter genera CI050Isolated strain from None WT Klebsiella genera CM002 Mutant of CI050Disruption of nifL gene ΔnifL::KanR SEQ ID with a kanamycin resistanceNO: 33 expression cassette (KanR) encoding the aminoglycoside O-phosphotransferase gene aph1 inserted. CM011 Mutant of CI019 Disruptionof nifL gene ΔnifL::SpecR SEQ ID with a spectinomycin NO: 34 resistanceexpression cassette (SpecR) encoding the streptomycin 3″-O-adenylyltransferase gene aadA inserted. CM013 Mutant of CI006 Disruptionof nifL gene ΔnifL::KanR SEQ ID with a kanamycin resistance NO: 35expression cassette (KanR) encoding the aminoglycoside O-phosphotransferase gene aph1 inserted. CM004 Mutant of CI010 Disruptionof amtB gene ΔamtB::KanR SEQ ID with a kanamycin resistance NO: 36expression cassette (KanR) encoding the aminoglycoside O-phosphotransferase gene aph1 inserted. CM005 Mutant of CI010 Disruptionof nifL gene ΔnifL::KanR SEQ ID with a kanamycin resistance NO: 37expression cassette (KanR) encoding the aminoglycoside O-phosphotransferase gene aph1 inserted. CM015 Mutant of CI006 Disruptionof nifL gene ΔnifL::Prm5 SEQ ID with a fragment of the NO: 38 regionupstream of the ompX gene inserted (Prm5). CM021 Mutant of CI006Disruption of nifL gene ΔnifL::Prm2 SEQ ID with a fragment of the NO: 39region upstream of an unannotated gene and the first 73 bp of that geneinserted (Prm2). CM023 Mutant of CI006 Disruption of nifL geneΔnifL::Prm4 SEQ ID with a fragm ent of the NO: 40 region upstream of theacpP gene and the first 121 bp of the acpP gene inserted (Prm4). CM014Mutant of CI006 Disruption of nifL gene ΔnifL::Prm1 SEQ ID with afragment of the NO: 41 region upstream of the lpp gene and the first 29bp of the lpp gene inserted (Prm1). CM016 Mutant of CI006 Disruption ofnifL gene ΔnifL::Prm9 SEQ ID with a fragment of the NO: 42 regionupstream of the lexA 3 gene and the first 21 bp of the lexA 3 geneinserted (Prm9). CM022 Mutant of CI006 Disruption of nifL geneΔnifL::Prm3 SEQ ID with a fragment of the NO: 43 region upstream of themntP 1 gene and the first 53 bp of the mntP 1 gene inserted (Prm3).CM024 Mutant of CI006 Disruption of nifL gene ΔnifL::Prm7 SEQ ID with afragment of the NO: 44 region upstream of the sspA gene inserted (Prm7).CM025 Mutant of CI006 Disruption of nifL gene ΔnifL::Prm10 SEQ ID with afragment of the NO: 45 region upstream of the hisS gene and the first 52bp of the hisS gene inserted (Prm10). CM006 Mutant of CI010 Disruptionof glnB gene ΔglnB::KanR SEQ ID with a kanamycin resistance NO: 46expression cassette (KanR) encoding the aminoglycoside O-phosphotransferase gene aph1 inserted. CM017 Mutant of CI028 Disruptionof nifL gene ΔnifL::KanR SEQ ID with a kanamycin resistance NO: 47expression cassette (KanR) encoding the aminoglycoside O-phosphotransferase gene aph1 inserted. CM011 Mutant of CI019 Disruptionof nifL gene ΔnifL::SpecR SEQ ID with a spectinomycin NO: 48 resistanceexpression cassette (SpecR) encoding the streptomycin 3″-O-adenylyltransferase gene aadA inserted. CM013 Mutant of CI006 Disruptionof nifL gene ΔnifL::KanR SEQ ID with a kanamycin resistance NO: 49expression cassette (KanR) encoding the aminoglycoside O-phosphotransferase gene aph1 inserted. CM005 Mutant of CI010 Disruptionof nifL gene ΔnifL::KanR SEQ ID with a kanamycin resistance NO: 50expression cassette (KanR) encoding the aminoglycoside O-phosphotransferase gene aph1 inserted. CM014 Mutant of CI006 Disruptionof nifL gene ΔnifL::Prm1 SEQ ID with a fragment of the NO: 51 regionupstream of the lpp gene and the first 29 bp of the lpp gene inserted(Prm1). CM015 Mutant of CI006 Disruption of nifL gene ΔnifL::Prm5 SEQ IDwith a fragment of the NO: 52 region upstream of the ompX gene inserted(Pnn5). CM023 Mutant of CI006 Disruption of nifL gene ΔnifL::Prm4 SEQ IDwith a fragment of the NO: 53 region upstream of the acpP gene and thefirst 121 bp of the acpP gene inserted (Prm4). CM029 Mutant of CI006Disruption of nifL gene ΔnifL::Prm5 SEQ ID SEQ ID with a fragment of theΔglnE- NO: 54 NO: 61 region upstream of the AR_KO1 ompX gene inserted(Prm5) and deletion of the 1287 bp after the start codon of the glnEgene containing the adenylyl-removing domain of glutamate-ammonia-ligase adenylyltransferase (ΔglnE-AR_KO1). CM014 Mutant of CI006Disruption of nifL gene ΔnifL::Prm1 SEQ ID with a fragment of the NO: 55region upstream of the lpp gene and the first 29 bp of the lpp geneinserted (Prm1). CM011 Mutant of CI019 Disruption of nifL geneΔnifL::SpecR SEQ ID with a spectinomycin NO: 56 resistance expressioncassette (SpecR) encoding the streptomycin 3″-O- adenylyltransferasegene aadA inserted. CM011 Mutant of CI019 Disruption of nifL geneΔnifL::SpecR SEQ ID with a spectinomycin NO: 57 resistance expressioncassette (SpecR) encoding the streptomycin 3″-O- adenylyltransferasegene aadA inserted. CM013 Mutant of CI006 Disruption of nifL geneΔnifL::KanR SEQ ID with a kanamycin resistance NO: 58 expressioncassette (KanR) encoding the aminoglycoside O- phosphotransferase geneaph1 inserted. CM011 Mutant of CI019 Disruption of nifL geneΔnifL::SpecR SEQ ID with a spectinomycin NO: 59 resistance expressioncassette (SpecR) encoding the streptomycin 3″-O- adenylyltransferasegene aadA inserted. CM011 Mutant of CI019 Disruption of nifL geneΔnifL::SpecR SEQ ID with a spectinomycin NO: 60 resistance expressioncassette (SpecR) encoding the streptomycin 3″-O- adenylyltransferasegene aadA inserted.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the disclosure and are not meant to limit the presentdisclosure in any fashion. Changes therein and other uses which areencompassed within the spirit of the disclosure, as defined by the scopeof the claims, will be recognized by those skilled in the art.

Example 1: Guided Microbial Remodeling—a Platform for the RationalImprovement of Microbial Species for Agriculture

An example overview of an embodiment of the Guided Microbial Remodeling(GMR) platform can be summarized in the schematic of FIG. 1A.

FIG. 1A illustrates that the composition of the microbiome can first becharacterized and a species of interest is identified (e.g. to find amicrobe with the appropriate colonization characteristics).

The metabolism of the species of interest can be mapped and linked togenetics. For example, the nitrogen fixation pathway of the microbe canbe characterized. The pathway that is being characterized can beexamined under a range of environmental conditions. For example, themicrobe's ability to fix atmospheric nitrogen in the presence of variouslevels of exogenous nitrogen in its environment can be examined. Themetabolism of nitrogen can involve the entrance of ammonia (NH₄ ⁺) fromthe rhizosphere into the cytosol of the bacteria via the AmtBtransporter. Ammonia and L-glutamate (L-Glu) are catalyzed by glutaminesynthetase and ATP into glutamine. Glutamine can lead to the formationof bacterial biomass and it can also inhibit expression of the nifoperon, i.e. it can be a competing force when one desires the microbe tofix atmospheric nitrogen and excrete ammonia. The nitrogen fixationpathway is characterized in great detail in earlier sections of thespecification.

Afterwards, a targeted non-intergeneric genomic alteration can beintroduced to the microbe's genome, using methods including, but notlimited to: conjugation and recombination, chemical mutagenesis,adaptive evolution, and gene editing. The targeted non-intergenericgenomic alteration can include an insertion, disruption, deletion,alteration, perturbation, modification, etc. of the genome.

Derivative remodeled microbes, which comprise the desired phenotyperesulting from the remodeled underlying genotype, are then used toinoculate crops.

The present disclosure provides, in certain embodiments,non-intergeneric remodeled microbes that are able to fix atmosphericnitrogen and supply such nitrogen to a plant. In aspects, thesenon-intergeneric remodeled microbes are able to fix atmosphericnitrogen, even in the presence of exogenous nitrogen.

FIG. 1B depicts an expanded view of the measurement of the microbiomestep. In some embodiments, the present disclosure finds microbialspecies that have desired colonization characteristics, and thenutilizes those species in the subsequent remodeling process.

The aforementioned Guided Microbial Remodeling (GMR) platform will nowbe described with more specificity.

In aspects, the GMR platform comprises the following steps:

-   -   A. Isolation—Obtain microbes from the soil, rhizosphere,        surface, etc. of a crop plant of interest;    -   B. Characterization—Involves characterizing the isolated        microbes for genotype/phenotypes of interest (e.g. genome        sequence, colonization ability, nitrogen fixation activity,        solubilization of P ability, excretion of a metabolite of        interest, excretion of a plant promoting compound, etc.)    -   C. Domestication—Development of a molecular protocol for        non-intergeneric genetic modification of the microbe;    -   D. Non-Intergeneric Engineering Campaign and        Optimization—Generation of derivative non-intergeneric microbial        strains with genetic modifications in key pathways (e.g.        colonization associated genes, nitrogen fixation/assimilation        genes, P solubilization genes);    -   E. Analytics—Evaluation of derived non-intergeneric strains for        phenotypes of interest both in vitro (e.g., ARA assays) and in        planta (e.g. colonization assays).    -   F. Iterate Engineering Campaign/Analytics—Iteration of steps D        and E for further improvement of microbial strain.

Each of the GMR platform process steps will now be elaborated uponbelow.

A. Isolation of Microbes

1. Obtain a Soil Sample

Microbes will be isolated from soil and/or roots of a plant. In oneexample, plants will be grown in a laboratory or a greenhouse in smallpots. Soil samples will be obtained from various agricultural areas. Forexample, soils with diverse texture characteristics can be collected,including loam (e.g. peaty clay loam, sandy loam), clay soil (e.g. heavyclay, silty clay), sandy soil, silty soil, peaty soil, chalky soil, andthe like.

2. Grow Bait Plants

Seeds of a bait plant (a plant of interest) (e.g. corn, wheat, rice,sorghum, millet, soybean, vegetables, fruits, etc.) will be planted intoeach soil type. In one example, different varieties of a bait plant willbe planted in various soil types. For example, if the plant of interestis corn, seeds of different varieties of corn such as field corn, sweetcorn, heritage corn, etc. will be planted in various soil typesdescribed above.

3. Harvest Soil and/or Root Samples and Plate on Appropriate Medium

Plants will be harvested by uprooting them after a few weeks (e.g. 2-4weeks) of growth. Alternative to growing plants in alaboratory/greenhouse, soil and/or roots of the plant of interest can becollected directly from the fields with different soil types.

To isolate rhizosphere microbes and epiphytes, plants will be removedgently by saturating the soil with distilled water or gently looseningthe soil by hand to avoid damage to the roots. If larger soil particlesare present, these particles will be removed by submerging the roots ina still pool of distilled water and/or by gently shaking the roots. Theroot will be cut and a slurry of the soil sticking to the root will beprepared by placing the root in a plate or tube with small amount ofdistilled water and gently shaking the plate/tube on a shaker orcentrifuging the tube at low speed. This slurry will be processed asdescribed below.

To isolate endophytes, excess soil on root surfaces will be removed withdeionized water. Following soil removal, plants will be surfacesterilized and rinsed vigorously in sterile water. A cleaned, 1 cmsection of root will be excised from the plant and placed in a phosphatebuffered saline solution containing 3 mm steel beads. A slurry will begenerated by vigorous shaking of the solution with a Qiagen TissueLyserII.

The soil and/or root slurry can be processed in various ways dependingon the desired plant-beneficial trait of microbes to be isolated. Forexample, the soil and root slurry can be diluted and inoculated ontovarious types of screening media to isolate rhizospheric, endophytic,epiphytic, and other plant-associated microbes. For example, if thedesired plant-beneficial trait is nitrogen fixation, then the soil/rootslurry will be plated on a nitrogen free media (e.g. Nfb agar media) toisolate nitrogen fixing microbes. Similarly, to isolate phosphatesolubilizing bacteria (PSB), media containing calcium phosphate as thesole source of phosphorus can be used. PSB can solubilize calciumphosphate and assimilate and release phosphorus in higher amounts. Thisreaction is manifested as a halo or a clear zone on the plate and can beused as an initial step for isolating PSB.

4. Pick Colonies, Purify Cultures, and Screen for the Presence of Genesof Interest

Populations of microbes obtained in step A3 are streaked to obtainsingle colonies (pure cultures). A part of the pure culture isresuspended in a suitable medium (e.g. a mixture of R2A and glycerol)and subjected to PCR analysis to screen for the presence of one or moregenes of interest. For example, to identify nitrogen fixing bacteria(diazotrophs), purified cultures of isolated microbes can be subjectedto a PCR analysis to detect the presence of nif genes that encodeenzymes involved in the fixation of atmospheric nitrogen into a form ofnitrogen available to living organisms.

5. Bank a Purified Culture

Purified cultures of isolated strains will be stored, for example at−80° C., for future reference and analysis.

B. Characterization of Isolated Microbes

1. Phylogenetic Characterization and Whole Genome Sequencing

Isolated microbes will be analyzed for phylogenetic characterization(assignment of genus and species) and the whole genome of the microbeswill be sequenced.

For phylogenetic characterization, 16S rDNA of the isolated microbe willbe sequenced using degenerate 16S rDNA primers to generate phylogeneticidentity. The 16S rDNA sequence reads will be mapped to a database toinitially assign the genus, species and strain name for isolatedmicrobes. Whole genome sequencing is used as the final step to assignphylogenetic genus/species to the microbes.

The whole genome of the isolated microbes will be sequenced to identifykey pathways. For the whole genome sequencing, the genomic DNA will beisolated using a genomic DNA isolation kit (e.g. QIAmp DNA mini kit fromQIAGEN) and a total DNA library will be prepared using the methods knownin the art. The whole genome will be sequenced using high throughputsequencing (also called Next Generation Sequencing) methods known in theart. For example, Illumina, Inc., Roche, and Pacific Biosciences providewhole genome sequencing tools that can be used to prepare total DNAlibraries and perform whole genome sequencing.

The whole genome sequence for each isolated strain will be assembled;genes of interest will be identified; annotated; and noted as potentialtargets for remodeling. The whole genome sequences will be stored in adatabase.

2. Assay the Microbe for Colonization of a Host Plant in a Greenhouse

Isolated microbes will be characterized for the colonization of hostplants in a greenhouse. For this, seeds of the desired host plant (e.g.,corn, wheat, rice, sorghum, and soybean) will be inoculated withcultures of isolated microbes individually or in combination and plantedinto soil. Alternatively, cultures of isolated microbes, individually orin combination, can be applied to the roots of the host plant byinoculating the soil directly over the roots. The colonization potentialof the microbes will be assayed, for example, using a quantitative PCR(qPCR) method described in a greater detail below.

3. Assay the Microbe for Colonization of the Host Plant in Small-ScaleField Trials and Isolate RNA from Colonized Root Samples (CAT Trials)

Isolated microbes will be assessed for colonization of the desired hostplant in small-scale field trials. Additionally, RNA will be isolatedfrom colonized root samples to obtain transcriptome data for the strainin a field environment. These small-scale field trials are referred toherein as CAT (Colonization and Transcript) trials, as these trialsprovide Colonization and Transcript data for the strain in a fieldenvironment.

For these trials, seeds of the host plant (e.g., corn, wheat, rice,sorghum, and soybean) will be inoculated using cultures of isolatedmicrobes individually or in combination and planted into soil.Alternatively, cultures of isolated microbes, individually or incombination, can be applied to the roots of the host plant byinoculating the soil directly over the roots. The CAT trials can beconducted in a variety of soils and/or under various temperature and/ormoisture conditions to assess the colonization potential and obtaintranscriptome profile of the microbe in various soil types andenvironmental conditions.

Colonization of roots of the host plant by the inoculated microbe(s)will be assessed, for example, using a qPCR method as described below.

In one protocol, the colonization potential of isolated microbes wasassessed as follows. One day after planting of corn seeds, 1 ml ofmicrobial overnight culture (SOB media) was drenched right at the spotof where the seed was located. 1 mL of this overnight culture wasroughly equivalent to about 10{circumflex over ( )}9 cfu, varying within3-fold of each other, depending on which strain is being used. Eachseedling was fertilized 3× weekly with 50 mL modified Hoagland'ssolution supplemented with either 2.5 mM or 0.25 mM ammonium nitrate. Atfour weeks after planting, root samples were collected for DNAextraction. Soil debris were washed away using pressurized water spray.These tissue samples were then homogenized using QIAGEN Tissuelyzer andthe DNA was then extracted using QIAmp DNA Mini Kit (QIAGEN) accordingto the recommended protocol. qPCR assay was performed using StratageneMx3005P RT-PCR on these DNA extracts using primers that were designed(using NCBI's Primer BLAST) to be specific to a loci in each of themicrobe's genome.

The presence of the genome copies of the microbe was quantified, whichreflected the colonization potential of the microbe. Identity of themicrobial species was confirmed by sequencing the PCR amplificationproducts.

Additionally, RNA will be isolated from colonized root and/or soilsamples and sequenced.

Unlike the DNA profile, an RNA profile varies depending on theenvironmental conditions. Therefore, sequencing of RNA isolated fromcolonized roots and/or soil will reflect the transcriptional activity ofgenes in planta in the rhizosphere.

RNA can be isolated from colonized root and/or soil samples at differenttime points to analyze the changes in the RNA profile of the colonizedmicrobe at these time points.

For example, RNA can be isolated from colonized root and/or soil samplesright after fertilization of the field and a few weeks afterfertilization of the field and sequenced to generate correspondingtranscriptional profile.

Similarly, RNA sequencing can be carried out under high phosphate andlow phosphate conditions to understand which genes are transcriptionallyactive or repressed under these conditions.

Methods for transcriptomic/RNA sequencing are known in the art. Briefly,total RNA will be isolated from the purified culture of the isolatedmicrobe; cDNA will be prepared using reverse transcriptase; and the cDNAwill be sequenced using high throughput sequencing tools describedabove.

Sequencing reads from the transcriptome analysis can be mapped to thegenomic sequence and transcriptional promoters for the genes of interestcan be identified.

4. Assay the Plant-Beneficial Activity of Isolated Microbes

The plant-beneficial activity of isolated microbes will be assessed.

For example, nitrogen fixing microbes will be assayed for nitrogenfixation activity using an acetylene reduction assay (ARA) or phosphatesolubilizing microbes will be assayed for phosphate solubilization. Anyparameter of interest can be utilized and an appropriate assay developedfor such. For instance, assays could include growth curves forcolonization metrics and assays for production of phytohormones likeindole acetic acid (IAA) or gibberellins. An assay for anyplant-beneficial activity that is of interest can be developed.

This step will confirm the phenotype of interest and eliminate any falsepositives.

5. Selection of Potential Candidates from Isolated Microbes

The data generated in the above steps will be used to select microbesfor further development. For example, microbes showing a desiredcombination of colonization potential, plant-beneficial activity, and/orrelevant DNA and RNA profile will be selected for domestication andremodeling.

C. Domestication of Selected Microbes

The selected microbes will be domesticated; wherein, the microbes willbe converted to a form that is genetically tractable and identifiable.

1. Test for Antibiotic Sensitivity

One way to domesticate the microbes is to engineer them with antibioticresistance. For this, the wild type microbial strain will be tested forsensitivity to various antibiotics. If the strain is sensitive to theantibiotic, then the antibiotic can be a good candidate for use ingenetic tools/vectors for remodeling the strain.

2. Design and Build a Vector

Vectors that are conditional for their replication (e.g. a suicideplasmid) will be constructed to domesticate the selected microbes (hostmicrobes). For example, a suicide plasmid containing an appropriateantibiotic resistance marker, a counter selectable marker, an origin ofreplication for maintenance in a donor microbe (e.g. E. coli), a geneencoding a fluorescent protein (GFP, RFP, YFP, CFP, and the like) toscreen for insertion through fluorescence, an origin of transfer forconjugation into the host microbe, and a polynucleotide sequencecomprising homology arms to the host genome with a desired geneticvariation will be constructed. The vector may comprise a SceI site andother additional elements.

Exemplary antibiotic resistance markers include ampicillin resistancemarker, kanamycin resistance marker, tetracycline resistance marker,chloramphenicol resistance marker, erythromycin resistance marker,streptomycin resistance marker, spectinomycin resistance marker, etc.Exemplary counter selectable markers include sacB, rpsL, tetAR, pheS,thyA, lacY, gata-1, ccdB, etc.

3. Generation of Donor Microbes

In one protocol, a suicide plasmid containing an appropriate antibioticresistance marker, a counter selectable marker, the λpir origin ofreplication for maintenance in E. coli ST18 containing the pirreplication initiator gene, a gene encoding green fluorescent protein(GFP) to screen for insertion through fluorescence, an origin oftransfer for conjugation into the host microbe, and a polynucleotidesequence comprising homology arms to the host genome with a desiredgenetic variation (e.g. a promoter from within the microbe's own genomefor insertion into a heterologous location) will be transformed into E.coli ST18 (an auxotroph for aminolevulinic acid, ALA) to generate donormicrobes.

4. Mix Donor Microbes with Host Microbes

Donor microbes will be mixed with host microbes (selected candidatemicrobes from step B5) to allow conjugative integration of the plasmidinto the host genome. The mixture of donor and host microbes will beplated on a medium containing the antibiotic and not containing ALA. Thesuicide plasmid is able to replicate in donor microbes (E. coli ST18),but not in the host. Therefore, when the mixture containing donor andhost microbes is plated on a medium containing the antibiotic and notcontaining ALA, only host cells that integrated the plasmid into itsgenome will be able to grow and form colonies on the medium. The donormicrobes will not grow due to the absence of ALA.

5. Confirm Integration of the Vector

A proper integration of the suicide plasmid containing the fluorescentprotein marker, the antibiotic resistance marker, the counter selectablemarker, etc. at the intended locus of the host microbe will be confirmedthrough fluorescence of colonies on the plate and using colony PCR.

6. Streak Confirm Integration Colony

A second round of homologous recombination in the host microbes willloop out (remove) the plasmid backbone leaving the desired geneticvariation (e.g. a promoter from within the microbe's own genome forinsertion into a heterologous location) integrated into the host genomeof a certain percentage of host microbes, while reverting a certainpercentage back to wild type.

Colonies of host microbes that have looped out the plasmid backbone (andtherefore, looped out the counter selectable marker) can be selected bygrowing them on an appropriate medium.

For example, if sacB is used as a counter selectable marker, loss ofthis marker due to the loss of the plasmid backbone will be tested bygrowing the colonies on a medium containing sucrose (sacB conferssensitivity to sucrose). Colonies that grow on this medium would havelost the sacB marker and the plasmid backbone and would either containthe desired genetic variation or be reverted to wild type. Also, thesecolonies will not fluoresce on the plate due to the loss of thefluorescent protein marker.

In some isolates, the sacB or other counterselectable markers do notconfer full sensitivity to sucrose or other counterselection mechanisms,which necessitates screening large numbers of colonies to isolate asuccessful loop-out. In those cases, loop-out may be aided by use of a“helper plasmid” that replicates independently in the host cell andexpresses a restriction endonuclease, e.g. SceI, which recognizes a sitein the integrated suicide plasmid backbone. The strain with theintegrated suicide plasmid is transformed with the helper plasmidcontaining an antibiotic resistance marker, an origin of replicationcompatible with the host strain, and a gene encoding a restrictionendonuclease controlled by a constitutive or inducible promoter. Thedouble-strand break induced in the integrated plasmid backbone by therestriction endonuclease promotes homologous recombination to loop-outthe suicide plasmid. This increases the number of looped-out colonies onthe counterselection plate and decreases the number of colonies thatneed to be screened to find a colony containing the desired mutation.The helper plasmid is then removed from the strain by culture and serialpassaging in the absence of antibiotic selection for the plasmid. Thepassaged cultures are streaked for single colonies, colonies are pickedand screened for sensitivity to the antibiotic used for selection of thehelper plasmid, as well as absence of the plasmid confirmed by colonyPCR. Finally, the genome is sequenced and the absence of helper plasmidDNA is confirmed as described in D6.

7. Confirm Integration of the Genetic Variation Through Colony PCR

The colonies that grew better on the sucrose-containing medium (or otherappropriate media depending on the counter selectable marked used) willbe picked and the presence of the genetic variation at the intendedlocus will be confirmed by screening the colonies using colony PCR.

Although this example describes one protocol for domesticating themicrobe and introducing genetic variation into the microbe, one ofordinary skill in the art would understand that the genetic variationcan be introduced into the selected microbes using a variety of othertechniques known in the art such as: polymerase chain reactionmutagenesis, oligonucleotide-directed mutagenesis, saturationmutagenesis, fragment shuffling mutagenesis, homologous recombination,ZFN, TALENS, CRISPR systems (Cas9, Cpf1, etc.), chemical mutagenesis,and combinations thereof.

8. Iterate Upon Steps C2-C7

If any of the steps C2-C7 fail to provide the intended outcome, thesteps will be repeated to design an alternative vector that may comprisedifferent elements for facilitating incorporation of desired geneticvariations and markers into the host microbe.

9. Develop a Standard Operating Procedure (SOP)

Once the steps C2-C7 can be reproduced consistently for a given strain,the steps will be used to develop a standard operating procedure (SOP)for that strain and vector. This SOP can be used to improve otherplant-beneficial traits of the microbe.

D. Non-Intergeneric Engineering Campaign and Optimization

1. Identify Gene Targets for Optimization

Selected microbes will be engineered/remodeled to improve performance ofthe plant-beneficial activity. For this, gene targets for improving theplant-beneficial activity will be identified.

Gene targets can be identified in various ways. For example, genes ofinterest can be identified while annotating the genes from the wholegenome sequencing of isolated microbes. They can be identified through aliterature search. For example, genes involved in nitrogen fixation areknown in the literature. These known genes can be used as targets forintroducing genetic variations. Gene targets can also be identifiedbased on the RNA sequencing data obtained in the step B3 (small-scalefield trials for colonization) or by performing RNA sequencing describedin the step below.

2. Select Promoters for Promoter Swaps

A desired genetic variation for improving the plant-beneficial activitycan comprise promoter swapping, in which the native promoter for atarget gene is replaced with a stronger or weaker promoter (whencompared to the native promoter) from within the microbe's genome, ordifferently regulated promoter (e.g. a N-independent). If the expressionof a target gene increases the plant-beneficial activity (e.g., nifA,the expression of which enhances nitrogen fixation in microbes), thedesired promoter for promoter swapping is a stronger promoter (comparedto the native promoter of the target gene) that would further increasethe expression level of the target gene compared to the native promoter.If the expression of a target gene decreases the plant-beneficialactivity (e.g., nifL that downregulates nitrogen fixation), the desiredpromoter for promoter swapping is a weak promoter (compared to thenative promoter of the target gene) that would substantially decreasethe expression level of the target gene compared to the native promoter.Promoters can be inserted into genes to “knock-out” a gene's expression,while at the same time upregulating the expression of a downstream gene.

Promoters for promoter swapping can be selected based on the RNAsequencing data. For example, the RNA sequencing data can be used toidentify strong and weak promoters, or constitutively active vs.inducible promoters.

For example, to identify strong and weak promoters, or constitutivelyactive vs. inducible promoters, in the nitrogen fixation pathway,selected microbes will be cultured in vitro under nitrogen-depleted andnitrogen-replete conditions; RNA of the microbe will be isolated fromthese cultures; and sequenced.

In one protocol, the RNA profile of the microbe under nitrogen-depletedand nitrogen-replete conditions will be compared and active promoterswith a desired transcription level will be identified. These promoterscan be selected to swap a weak promoter.

Promoters can also be selected using the RNA sequencing data obtained inthe step B3 that reflects the RNA profile of the microbe in planta inthe host plant rhizosphere.

RNA sequencing under various conditions allows for selection ofpromoters that: a) are active in the rhizosphere during the host plantgrowth cycle in fertilized field conditions, and b) are also active inrelevant in vitro conditions so they can be rapidly screened.

In an exemplary protocol, in planta RNA sequencing data fromcolonization assays (e.g. step B3) is used to measure the expressionlevels of genes in isolated microbes. In one embodiment, the level ofgene expression is calculated as reads per kilobase per million mappedreads (RPKM). The expression level of various genes is compared to theexpression level of a target gene and at least the top 10, 20, 30, 40,50, 60, or 70 promoters, associated with the various genes, that showthe highest or lowest level of expression compared to the target geneare selected as possible candidates for promoter swapping. Thus, onelooks at expression levels of various genes relative to a target geneand then selects genes that demonstrate increased expression relative toa target (or standard) gene and then find the promoters associated withsaid genes.

For example, if the target gene is upregulation of nifA, the first 10,20, 30, 40, 50, or 60 promoters for genes that show the highest level ofexpression compared to nifA are selected as possible candidates forpromoter swapping.

These candidates can be further short-listed based on in vitro RNAsequencing data. For example, for nifA as the target gene, possiblepromoter candidates selected based on the in planta RNA sequencing dataare further selected by choosing promoters with similar or increasedgene expression levels compared to nifA under in vitro nitrogen-depletevs. nitrogen-replete conditions.

The set of promoters selected in this step are used to swap the nativepromoter of the target gene (e.g. nifA). Remodeled strains with swappedpromoters are tested in in vitro assays; strains with lower thanexpected activity are eliminated; and strains with expected or higherthan expected activity are tested in field. The cycle of promoterselection may be repeated on remodeled strains to further improve theirplant-beneficial activity.

Described here is an exemplary promoter swap experiment that was carriedout based on in planta and in vitro RNA sequencing data from Klebsiellavariicola strain, CI137 to improve the nitrogen fixation trait. CI137was analyzed in ARA assays at 0 mM and 5 mM glutamine concentration andRNA was extracted from these ARA samples. The RNA was sequenced viaNextSeq and a subset of reads from one sample was mapped to the CI137genome (in vitro RNA sequencing data). RNA was extracted from the rootsof corn plants at V5 stage in the colonization and activity assay (e.g.step B3) for CI137. Samples from 6 plants were pooled; the RNA from thepooled sample was sequenced using NextSeq, and reads were mapped to theCI137 genome (in planta RNA sequencing data). Out of 2×10⁸ total reads,7×10⁴ reads mapped to CI137. In planta RNA sequencing data was used torank genes in order of in planta expression levels and the expressionlevels were compared to the native nifA expression level. The first 40promoters that showed the highest expression level (based on geneexpression) compared to the native nifA expression level were selected.These 40 promoters were further short-listed based on the in vitro RNAsequencing data, where promoters with increased or similar in vitroexpression levels compared to nifA were selected. The final list ofpromoters included 17 promoters and 2 versions of most promoters wereused to generate promoter swap mutants; thus a total of 30 promoterswere tested. Generation of a suite of CI137 mutants where nifL wasdeleted partially or completely and the 30 promoters inserted(ΔnifL::Prm) was attempted. 28 out of 30 mutants were generatedsuccessfully. The ΔnifL::Prm mutants were analyzed in ARA assays at 0 mMand 5 mM glutamine concentration and RNA was extracted from these ARAsamples. Several mutants showed lower than expected or decreased ARAactivity compared to the WT CI137 strain. A few mutants showed higherthan expected ARA activity.

A person of ordinary skill in the art would appreciate from the aboveexample that while in planta and/or in vitro RNA sequencing data can beused to select promoters for promoter swapping, the step of promoterselection is highly unpredictable and involves many challenges.

For example, in planta RNA sequencing mainly reveals the genes that arehighly expressed; however, it is difficult to detect fine differences ingene expression and/or genes with low expression levels. For instance,in some in planta RNA sequencing experiments, only about 40 out of about5000 genes from a microbial genome were detected. Thus, in planta RNAsequencing technique is useful to identify abundantly expressed genesand their corresponding promoters; however, the technique has difficultyin identifying low expression genes and corresponding promoters andsmall differences between gene expression.

Furthermore, in planta RNA profile reflects the status of the genes atthe time the microbes were isolated; however, a slight change in thefield conditions can substantially change the RNA profile ofrhizosphere/epiphytic/endophytic microbes. Therefore, it is difficult topredict in advance whether the promoters selected based on one fieldtrial RNA sequencing data would provide desirable expression levels ofthe target gene when remodeled strains are tested in vitro and in field.

Additionally, in planta evaluation is time and resource-consuming;therefore, in planta experiments cannot be conducted often and/orrepeated quickly or easily. On the other hand, while in vitro RNAsequencing can be conducted relatively quickly and easily, the in vitroconditions do not mimic the field conditions and promoters that may showhigh activity in vitro may not show comparable activity in planta.

Moreover, promoters often don't behave as predicted in a new context.Therefore, in planta and in vitro RNA sequencing data can at best serveas a starting point in the step of promoter selection; however, arrivingat any particular promoter that would provide desirable expressionlevels of the target gene in the field is, in some instances,unpredictable.

Another limitation in the step of promoter selection is the number ofavailable promoters. Because one of the goals of the present inventionis to provide non-transgenic microbes; promoters for promoter swappingneed to be selected from within the microbe's genome, or genus. Thus,unlike a transgenic approach, the present process cannot merely go outinto the literature and find/use a well-characterized transgenicpromoter from a different host organism.

Another constraint is that the promoter must be active in planta duringa desired growth phase. For example, the highest requirement fornitrogen in plants is generally late in the growing season, e.g. latevegetative and early reproductive phases. For example, in corn, nitrogenuptake is the highest during V6 (6 leaves) through R1 (reproductivestage 1) stages. Therefore, to increase the availability of nitrogenduring V6 through R1 stages of corn, remodeled microbes must showhighest nitrogen fixation activity during these stages of the cornlifecycle. Accordingly, promoters that are active in planta during thelate vegetative and early reproductive stages of corn need to beselected. This constraint not only reduces the number of promoters thatmay be tested in promoter swapping, but also make the step of promoterselection unpredictable. As discussed above, unpredictability arises, inpart, because although the RNA sequencing data from small scale fieldtrials (e.g. step B3) may be used to identify promoters that are activein planta during a desired growth stage, the RNA data is based on thefield conditions (e.g., type of soil, level of water in the soil, levelof available nitrogen, etc.) at the time of sample collection. As one ofordinary skill in the art would understand, the field conditions maychange over the period of time within the same field and also changesubstantially across various fields. Thus, the promoters selected underone field condition may not behave as expected under other fieldconditions. Similarly, selected promoters may not behave as expectedafter swapping. Therefore, it is difficult to anticipate in advancewhether the selected promoters would be active in planta during adesired growth phase of a plant of interest.

3. Design Non-Intergeneric Genetic Variations

Based on steps D1 (identification of gene targets) and D2(identification of promoters for promoter swaps), non-intergenericgenetic variations will be designed.

The term “non-intergeneric” indicates that the genetic variation to beintroduced into the host does not contain a nucleic acid sequence fromoutside the host genus (i.e., no transgenic DNA). Although vectorsand/or other genetic tools will be used to introduce the geneticvariation into the host microbe, the methods of the present disclosureinclude steps to loop-out (remove) the backbone vector sequences orother genetic tools introduced into the host microbe leaving only thedesired genetic variation into the host genome. Thus, the resultingmicrobe is non-transgenic.

Exemplary non-intergeneric genetic variations include a mutation in thegene of interest that may improve the function of the protein encoded bythe gene; a constitutionally active promoter that can replace theendogenous promoter of the gene of interest to increase the expressionof the gene; a mutation that will inactivate the gene of interest; theinsertion of a promoter from within the host's genome into aheterologous location, e.g. insertion of the promoter into a gene thatresults in inactivation of said gene and upregulation of a downstreamgene; and the like. The mutations can be point mutations, insertions,and/or deletions (full or partial deletion of the gene). For example, inone protocol, to improve the nitrogen fixation activity of the hostmicrobe, a desired genetic variation may comprise an inactivatingmutation of the nifL gene (negative regulator of nitrogen fixationpathway) and/or comprise replacing the endogenous promoter of the nifHgene (nitrogenase iron protein that catalyzes a key reaction to fixatmospheric nitrogen) with a constitutionally active promoter that willdrive the expression of the nifH gene constitutively.

4. Generate Non-Intergeneric Derivative Strains

After designing the non-intergeneric genetic variations, steps C2-C7will be carried out to generate non-intergeneric derivative strains(i.e. remodeled microbes).

5. Bank a Purified Culture of the Remodeled Microbe

A purified culture of the remodeled microbe will be preserved in a bank,so that gDNA can be extracted for whole genome sequencing describedbelow.

6. Confirm Presence of the Desired Genetic Variation

The genomic DNA of the remodeled microbe will be extracted and the wholegenome sequencing will be performed on the genomic DNA using methodsdescribed previously. The resulting reads will be mapped to the readspreviously stored in LIMS to confirm: a) presence of the desired geneticvariation, and b) complete absence of reads mapping to vector sequences(e.g. plasmid backbone or helper plasmid sequence) that were used togenerate the remodeled microbe.

This step allows sensitive detection of non-host genus DNA (transgenicDNA) that may remain in the strain after looping out of the vectorbackbone (e.g. suicide plasmid) method and could provide a control foraccidental off-target insertion of the genetic variation, etc.

E. Analytics Upon Remodeled Microbes

1. Analysis of the Plant-Beneficial Activity

The plant-beneficial activity and growth kinetics of the remodeledmicrobes will be assessed in vitro.

For example, strains remodeled for improving nitrogen fixation functionwill be assessed for nitrogen fixation activity and fitness throughacetylene reduction assays, ammonium excretion assays, etc.

Strains remodeled for improved phosphate solubilization will be assessedfor the phosphate solubilization activity.

This step allows rapid, medium to high throughput screening of remodeledstrains for the phenotypes of interest.

2. Analysis of Colonization and Transcription of the Altered Genes

Remodeled strains will be assessed for colonization of the host planteither in the greenhouse or in the field using the steps described inB3. Additionally, RNA will be isolated from colonized root and/or soilsamples and sequenced to analyze the transcriptional activity of targetgenes. Target genes comprise the genes containing the genetic variationintroduced and may also comprise other genes that play a role in theplant-beneficial trait of the microbe.

For example, a cluster of genes, the nif genes, controls the nitrogenfixation activity of microbes. Using the protocol described above, agenetic variation may be introduced into one of the nif genes (e.g. apromoter insertion), whereas the other genes in the nif cluster are intheir endogenous form (i.e. their gene sequence and/or the promoterregion is not altered). The RNA sequencing data will be analyzed for thetranscriptional activity of the nif gene containing the geneticvariation and may also be analyzed for other nif genes that are notaltered directly, by the inserted genetic change, but nonetheless may beinfluenced by the introduced genetic change.

This step allows determination of the fitness of top in vitro performingstrains in the rhizosphere and allows measurement of the transcriptionalactivity of altered genes in planta.

F. Iterate Engineering Campaign/Analytics

The data from in vitro and in planta analytics (steps E1 and E2) will beused to iteratively stack beneficial mutations.

Furthermore, steps A-E described above may be repeated to fine-tune theplant-beneficial traits of the microbes. For example, plants will beinoculated using microbial strains remodeled in the first round;harvested after a few weeks of growth; and microbes from the soil and/orroots of the plants will be isolated. The functional activity(plant-beneficial trait and colonization potential) and the DNA and RNAprofile of isolated microbes will be characterized, in order to selectmicrobes with improved plant-beneficial activity and colonizationpotential. The selected microbes will be remodeled to further improvethe plant-beneficial activity. Remodeled microbes will be screened forthe functional activity (plant-beneficial trait and colonizationpotential) and RNA profile in vitro and in planta and the top performingstrains will be selected. If desired, steps A-E can be repeated tofurther improve the plant-beneficial activity of the remodeled microbesfrom the second round. The process can be repeated for 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more rounds.

The exemplary steps described above are summarized in Table A below.

TABLE A An Overview of an Embodiment of the Guided Microbial RemodelingPlatform Steps Contribution Alternate Forms A Isolation 1 Obtain a soilsample Provides WT soil microbes to be isolated 2 Grow corn “bait Allowsselection of plant- Wheat, sorghum, rice, millet, plants” in soil samplebeneficial microbes by soybean, etc. rhizosphere 3 Harvest, clean andDown-select soil microbes Other nitrogen-free media, other extract rootsample to those that a) colonize the selective or screening media (eg.and plate on nitrogen- root and b) fix atmospheric for phosphatesolubilization) free (specifically nitrogen NfB) media 4 Pick colonies,purify Down-select microbes to Degenerate primers for other cultures andscreen those containing the nifH genes of interest, e.g. ipdC forpresence of nifH gene (eliminate false- (phytohormone biosynthesis)using degenerate positive from media primers screen) 5 Bank a purifiedculture of the strain B Characterization 1 Sequence and Characterizegenome for assemble the genome key pathways of the strain using Illuminaand/or PacBio platform 2 Assay the microbe for Down-select for microbesWheat, sorghum, rice, millet, colonization of corn that colonize theplant well soybean, etc., other methods for roots in the assayingcolonization (e.g. greenhouse (qPCR- plating) based method) 3 Assay themicrobe for Known internally as “CAT” Larger field trials, other crops,colonization of com trials, these provide other methods for assayingroots in a small-scale Colonization And colonization (e.g. plating)field trials (qPCR- Transcript data for the based method) and strain ina field isolate RNA from environment colonized root samples 4 Assay themicrobe for Confirm N-fixation nitrogen fixation phenotype of strainactivity in an acetylene reduction assay (ARA) 5 Use the above data toAllows selection of select candidate greatest-potential microbe forfurther candidates domestication and optimization C Domestication 1 Testmicrobes for Determine which antibiotic sensitivity to various selectionmarkers can be antibiotics used to transform genetic tools 2 Design andbuild a These are the “parts” Plasmid could contain a SceI site suicideplasmid necessary to maintain the or other counter-selectable containingan plasmid and carry out marker, alternate fluorescent appropriateantibiotic conjugation, insertion and reporters, additional elementsresistance marker, “loop-out” of the host sacB counter- genomeselectable marker, origin of replication for maintenance in E. coli, GFPto screen for insertion through fluorescence, origin of transfer forconjugation into the host, homology arms to the host genome, and thedesired mutation. 3 Transform suicide Preparation for conjugation Coulduse a different donor strain plasmid into E. coli into host; plasmid ofE. coli or other microbe; ST18 (an auxotroph maintenance differentauxotrophic marker for aminolevulinic acid, ALA) to generate donor cells4 Mix donor cells with The suicide plasmid is able Could use a differentdonor strain recipient host cells to to replicate in E. coli but of E.coli or other microbe; conjugate, and plate not in the host Thereforedifferent auxotrophic marker on media selecting for plating of themixture on the antibiotic such plates means that only resistance markerand host cells that received the NOT containing ALA plasmid andexperience plasmid integration into the chromosome will be able to growand form colonies. The E coli ST18 is unable to grow due to the absenceof ALA 5 Confirm integration Confirms proper integration of the plasmidof the suicide plasmid through GFP backbone containing GFP,fluorescence, and the antibiotic resistance integration at the cassette,the sacB marker, intended locus etc. through colony PCR 6 Streakconfirmed The sacB marker confers Different counter selectableintegration colony on sensitivity to sucrose; marker, SceI-mediatedloop-out, a plate containing colonies which have etc. sucrose and screenfor undergone a second round non-fluorescent of homologous coloniesrecombination and “looped- out” the plasmid will grow better and notfluoresce on the plate. 7 Screen looped-out Upon the second colonies forthe homologous recombination intended mutation event only 50% of loopedusing colony PCR out colonies should contain the mutation, the other 50%will be WT 8 If any of the steps 2-7 Allows iterative fail, go back tostep 2 troubleshooting of suicide and re-design with plasmid to developa alternate plasmid working protocol parts 9 Once steps 2-7 can bereliably performed, develop an SOP for that strain/plasmid to be usedfor Optimization D Non-Intergeneric Engineering Campaign andOptimization 1 Identify gene targets for optimizing a pathway, eg. nifgenes through literature search 2 Select promoters for Allows forselection of Alternate crops; alternate RNAseq promoter swaps usingpromoters that a) are active data conditions (greenhouse, field, RNAseqdata in the rhizosphere during in vitro, whatever's relevant forcollected both in vitro the corn growth cycle in the phenotype targeted)in N-depleted and N- fertilized field conditions b) replete conditions,are also active in in vitro N- and in planta from replete conditions sothey the corn rhizosphere can be rapidly screened. (Collected in stepB3) 3 Design non- No DNA from outside the Alter regulatory sequences(e.g. intergeneric host chromosome is added, RBS), non-coding RNAs, etc.mutations in key therefore the resulting genes: deletions (full microbeis non-transgenic or partial gene), promoter swaps, or single base pairchanges; store these designs in our LIMS 4 Using the established Weperform this in higher protocol, carry out throughput than the stepsC2-7 to generate domestication step - up to non-intergeneric 20 or sostrains at once per derivative strains person. (mutants) 5 Bank apurified culture of the strain, extract gDNA and conduct WGS viaIllumina 6 Map the resulting Allows very sensitive Suicide plasmidremoval is fairly reads to the designs detection of non- reliable;however use of other stored in LIMS to intergeneric DNA that may stableplasmids in alternate confirm a) presence remain in the strain aftermethods necessitates this extra of the desire mutation the suicideplasmid method; step to ensure with complete and b) complete confirmabsence of confidence that no transgenic absence of reads transgenicDNA, controls DNA that was previously mapping to any for accidentaloff-target transformed in remains in the suicide plasmid or insertion ofthe suicide strain. other plasmid plasmid, etc. sequences used togenerate the strains E Analytics 1 Analyze the strains Allow rapid, med-to high- Any other in vitro assay, e.g. for in vitro nitrogen throughputscreening of phosphate solubilization, qPCR fixation activity andmutants for phenotypes of for transcription of specific genes, fitnessthrough ARA, interest etc. ammonium excretion assays, and growth curves2 Analyze the strains Measure fitness of top in for colonization vitroperforming strains in (qPCR) and the rhizosphere; measure transcriptionof target transcription of promoter- and promoter- swapped genes inplanta swapped genes (Nanostring) in the plant (greenhouse or field) FIterate Engineering Campaign/Analytics 1 Use data from in vitro and inplanta analytics to iteratively stack beneficial mutations.

Traditional Approaches to Creating Biologicals for Agriculture Sufferfrom Drawbacks Inherent in their Methodology

Unlike pure bioprospecting of wild type (WT) microbes or transgenicapproaches, GMR allows for non-intergeneric genetic optimization of keyregulatory networks within the microbe, which improves plant-beneficialphenotypes over WT microbes, but doesn't have the risks associated withtransgenic approaches (e.g. unpredictable gene function, public andregulatory concerns). See, FIG. 1C for a depiction of a problematic“traditional bioprospecting” approach, which has several drawbackscompared to the taught GMR platform.

Other methods for developing microbials for agriculture are focused oneither extensive lab development, which often fails at the field scale,or extensive greenhouse or “field-first” testing without anunderstanding of the underlying mechanisms/plant-microbe interactions.See, FIG. 1D for a depiction of a problematic “field-first approach tobioprospecting” system, which has several drawbacks compared to thetaught GMR platform.

The GMR Platform Solves these Problems in Numerous Ways

One strength of the GMR platform is the identification of activepromoters, which are active at key physiologically important times for atarget crop, and which are also active under particular, agriculturallyrelevant, environmental conditions.

As has been explained, within the context of nitrogen fixation, the GMRplatform is able to identify microbial promoter sequences, which areactive under environmental conditions of elevated exogenous nitrogen,which thereby allows the remodeled microbe to fix atmospheric nitrogenand deliver it to a target crop plant, under modern agricultural rowcrop conditions, and at a time when a plant needs the fixed nitrogen themost. See, FIG. 1E for a depiction of the time period in the corn growthcycle, at which nitrogen is needed most by the plant. The taught GMRplatform is able to create remodeled microbes that supply nitrogen to acorn plant at the time period in which the nitrogen is needed, and alsodeliver such nitrogen even in the presence of exogenous nitrogen in thesoil environment.

These promoters can be identified by rhizosphere RNA sequencing and readmapping to the microbe's genome sequence, and key pathways can be“reprogrammed” to be turned on or off during key stages of the plantgrowth cycle. Additionally, through whole genome sequencing of optimizedmicrobes and mapping to previously-transformed sequences, the method hasthe ability to ensure that no transgenic sequences are accidentallyreleased into the field through off-target insertion of plasmid DNA,low-level retention of plasmids not detected through PCR or antibioticresistance, etc.

The GMR platform combines these approaches by evaluating microbesiteratively in the lab and plant environment, leading to microbes thatare robust in greenhouse and field conditions rather than just in labconditions.

Various aspects and embodiments of the taught GMR platform can be foundin FIGS. 1F-1I. The GMR platform culminates in thederivation/creation/production of remodeled microbes that possess aplant-beneficial property, e.g. nitrogen fixation.

The traditional bioprospecting methods are not able to produce microbeshaving the aforementioned properties.

Properties of a Microbe Remodeled for Nitrogen Fixation

In the context of remodeling microbes for nitrogen fixation, there areseveral properties that the remodeled microbe may possess. For instance,FIG. 1J depicts 5 properties that can be possessed by remodeled microbesof the present disclosure.

Furthermore, as can be seen in Example 2, the present inventors haveutilized the GMR platform to produce remodeled non-intergeneric bacteria(i.e. Kosakonia sacchari) capable of fixing atmospheric nitrogen anddelivering said nitrogen to a corn plant, even under conditions in whichexogenous nitrogen is present in the environment. See, FIG. 1K-M, whichillustrate that the remodeling process successfully: (1) decoupled nifAexpression from endogenous nitrogen regulation; and (2) improved theassimilation and excretion of fixed nitrogen.

These remodeled microbes ultimately result in corn yield improvement,when applied to corn crops. See, FIG. 1N.

The GMR Platform Provides an Approach to Nitrogen Fixation and Deliverythat Solves Pressing Environmental Concerns

As explained previously, the nitrogen fertilizer produced by theindustrial Haber-Bosch process is not well utilized by the target crop.Rain, runoff, heat, volatilization, and the soil microbiome degrade theapplied chemical fertilizer. This equates to not only wasted money, butalso adds to increased pollution instead of harvested yield. To thisend, the United Nations has calculated that nearly 80% of fertilizer islost before a crop can utilize it. Consequently, modern agriculturalfertilizer production and delivery is not only deleterious to theenvironment, but it is extremely inefficient. See, FIG. 1O, illustratingthe inefficiency of current nitrogen delivery systems, which result inunderfertilized fields, over fertilized fields, and environmentallydeleterious nitrogen runoff.

The current GMR platform, and resulting remodeled microbes, provide abetter approach to nitrogen fixation and delivery to plants. As will beseen in the below Examples, the non-intergeneric remodeled microbes ofthe disclosure are able to colonize the roots of a corn plant and spoonfeed said corn plants with fixed atmospheric nitrogen, even in thepresence of exogenous nitrogen. This system of nitrogen fixation anddelivery—enabled by the taught GMR platform—will help transform modernagricultural to a more environmentally sustainable system.

Example 2: Guided Microbial Remodeling—an Example Embodiment for theRational Improvement of Nitrogen Fixation

A diversity of nitrogen fixing bacteria can be found in nature,including in agricultural soils. However, the potential of a microbe toprovide sufficient nitrogen to crops to allow decreased fertilizer usemay be limited by repression of nitrogenase genes in fertilized soils aswell as low abundance in close association with crop roots.Identification, isolation and breeding of microbes that closelyassociate with key commercial crops might disrupt and improve theregulatory networks linking nitrogen sensing and nitrogen fixation andunlock significant nitrogen contributions by crop-associated microbes.To this end, nitrogen fixing microbes that associate with and colonizethe root system of corn were identified. This step corresponds to the“Measure the Microbiome Composition” depicted in FIG. 1A and FIG. 1B.

Root samples from corn plants grown in agronomically relevant soils werecollected, and microbial populations extracted from the rhizosphere andendosphere. Genomic DNA from these samples was extracted, followed by16S amplicon sequencing to profile the community composition.

A Kosakonia sacchari microbe (strain PBC6.1) was isolated and classifiedthrough 16S rRNA and whole genome sequencing. This is a particularlyinteresting nitrogen fixer capable of colonizing to nearly 21% abundanceof the root-associated microbiota (FIG. 2). To assess strain sensitivityto exogenous nitrogen, nitrogen fixation rates in pure culture weremeasured with the classical acetylene reduction assay (ARA) and varyinglevels of glutamine supplementation. The species exhibited a high levelof nitrogen fixing activity in nitrogen-free media, yet exogenous fixednitrogen repressed nif gene expression and nitrogenase activity (StrainPBC6.1, FIG. 3C, FIG. 3D). Additionally, when released ammonia wasmeasured in the supernatant of PBC6.1 grown in nitrogen-fixingconditions, very little release of fixed nitrogen could be detected(FIG. 3E).

We hypothesized that PBC6.1 could be a significant contributor of fixednitrogen in fertilized fields if regulatory networks controllingnitrogen metabolism were remodeled to allow optimal nitrogenaseexpression and ammonia release in the presence of fixed nitrogen.

Sufficient genetic diversity should exist within the PBC6.1 genome toenable broad phenotypic remodeling (as a result of remodeling theunderlying genetic architecture in a non-intergeneric manner) withoutthe insertion of transgenes or synthetic regulatory elements. Theisolated strain has a genome of at least 5.4 Mbp and a canonicalnitrogen fixation gene cluster. Related nitrogen metabolism pathways inPBC6.1 are similar to those of the model organism for nitrogen fixation,Klebsiella oxytoca m5al.

Several gene regulatory network nodes were identified which may augmentnitrogen fixation and subsequent transfer to a host plant, particularlyin high exogenous concentrations of fixed nitrogen (FIG. 3A). The nifLAoperon directly regulates the rest of the nif cluster throughtranscriptional activation by NifA and nitrogen- and oxygen-dependentrepression of NifA by NifL. Disruption of nifL can abolish inhibition ofNifA and improve nif expression in the presence of both oxygen andexogenous fixed nitrogen. Furthermore, expressing nifA under the controlof a nitrogen-independent promoter may decouple nitrogenase biosynthesisfrom regulation by the NtrB/NtrC nitrogen sensing complex.

The assimilation of fixed nitrogen by the microbe to glutamine byglutamine synthetase (GS) is reversibly regulated by the two-domainadenylyltransferase (ATase) enzyme GlnE through the adenylylation anddeadenylation of GS to attenuate and restore activity, respectively.Truncation of the GlnE protein to delete its adenylyl-removing (AR)domain may lead to constitutively adenylated glutamine synthetase,limiting ammonia assimilation by the microbe and increasing intra- andextracellular ammonia.

Finally, reducing expression of AmtB, the transporter responsible foruptake of ammonia, could lead to greater extracellular ammonia.

To generate rationally designed microbial phenotypes without the use oftransgenes, two approaches were employed to remodel the underlyinggenetic architecture of the microbe: (1) creating markerless deletionsof genomic sequences encoding protein domains or whole genes, and (2)rewiring regulatory networks by intragenomic promoter rearrangement.

Through an iterative remodeling process, several non-transgenicderivative strains of PBC6.1 were generated (Table 25).

TABLE 25 List of isolated and derivative K. sacchari strains used inthis work. Prm, promoter sequence derived from the PBC6.1 genome;ΔglnE_(AR)1 and ΔglnE_(AR)2, different truncated versions of glnE generemoving the adenylyl-removing domain sequence. Strain ID GenotypePBC6.1 WT PBC6.14 ΔnifL::Prm1 PBC6.15 ΔnifL::Prm5 PBC6.22 ΔnifL::Prm3PBC6.37 ΔnifL::Prm1 ΔglnE_(AR)2 PBC6.38 ΔnifL::Prm1 ΔglnE_(AR)1 PBC6.93ΔnifL::Prm1 ΔglnE_(AR)2 ΔamtB PBC6.94 ΔnifL::Prm1 ΔglnE_(AR)1 ΔamtB

Several in vitro assays were performed to characterize specificphenotypes of the derivative strains. The ARA was used to assess strainsensitivity to exogenous nitrogen, in which PBC6.1 exhibited repressionof nitrogenase activity at high glutamine concentrations (FIG. 3D). Incontrast, most derivative strains showed a derepressed phenotype withvarying levels of acetylene reduction observed at high glutamineconcentrations. Transcriptional rates of nifA in samples analyzed byqPCR correlated well with acetylene reduction rates (FIG. 4), supportingthe hypothesis that nifL disruption and insertion of anitrogen-independent promoter to drive nifA can lead to nif clusterderepression.

Strains with altered GlnE or AmtB activity showed markedly increasedammonium excretion rates compared to wild type or derivative strainswithout these mutations (FIG. 3E), illustrating the effect of thesegenotypes on ammonia assimilation and reuptake.

Two experiments were performed to study the interaction of PBC6.1derivatives (remodeled microbes) with corn plants and quantifyincorporation of fixed nitrogen into plant tissues. First, rates ofmicrobial nitrogen fixation were quantified in a greenhouse study usingisotopic tracers. Briefly, plants are grown with 15N labeled fertilizer,and diluted concentrations of 15N in plant tissues indicatecontributions of fixed nitrogen from microbes. Corn seedlings wereinoculated with selected microbial strains, and plants were grown to theV6 growth stage. Plants were subsequently deconstructed to enablemeasurement of microbial colonization and gene expression as well asmeasurement of 15N/14N ratios in plant tissues by isotope ratio massspectrometry (IRMS). Analysis of the aerial tissue showed a small,nonsignificant contribution by PBC6.38 to plant nitrogen levels, and asignificant contribution by PBC6.94 (p=0.011). Approximately 20% of thenitrogen found in above-ground corn leaves was produced by PBC6.94, withthe remainder coming from the seed, potting mix, or “background”fixation by other soilborne microbes (FIG. 5C). This illustrates thatour microbial breeding and remodeling pipeline can generate remodeledstrains capable of making significant nitrogen contributions to plantsin the presence of nitrogen fertilizer. Microbial transcription withinplant tissues was measured, and expression of the nif gene cluster wasobserved in derivative remodeled strains, but not the wild type strain(FIG. 5B), showing the importance of nif derepression for contributionof BNF to crops in fertilized conditions. Root colonization measured byqPCR demonstrated that colonization density is different for each of thestrains tested (FIG. 5A). A 50-fold difference in colonization wasobserved between PBC6.38 and PBC6.94. This difference could be anindication that PBC6.94 has reduced fitness in the rhizosphere relativeto PBC6.38 as a result of high levels of fixation and excretion.

Methods Media

Minimal medium contains (per liter) 25 g Na2HPO₄, 0.1 g CaCL₂-2H₂O, 3 gKH₂PO₄, 0.25 g MgSO₄.7H₂O, 1 g NaCl, 2.9 mg FeCl₃, 0.25 mg Na₂MoO₄.2H₂O,and 20 g sucrose. Growth medium is defined as minimal mediumsupplemented with 50 ml of 200 mM glutamine per liter.

Isolation of Diazotrophs

Corn seedlings were grown from seed (DKC 66-40, DeKalb, Ill.) for twoweeks in a greenhouse environment controlled from 22° C. (night) to 26°C. (day) and exposed to 16 hour light cycles in soil collected from SanJoaquin County, CA. Roots were harvested and washed with steriledeionized water to remove bulk soil. Root tissues were homogenized with2 mm stainless steel beads in a tissue lyser (TissueLyser II, Qiagen P/N85300) for three minutes at setting 30, and the samples were centrifugedfor 1 minute at 13,000 rpm to separate tissue from root-associatedbacteria. Supernatants were split into two fractions, and one was usedto characterize the microbiome through 16S rRNA amplicon sequencing andthe remaining fraction was diluted and plated on Nitrogen-free Broth(NfB) media supplemented with 1.5% agar. Plates were incubated at 30° C.for 5-7 days. Colonies that emerged were tested for the presence of thenifH gene by colony PCR with primers Ueda19f and Ueda406r. Genomic DNAfrom strains with a positive nifH colony PCR was isolated (QIAamp DNAMini Kit, Cat No. 51306, QIAGEN, Germany) and sequenced (Illumina MiSeqv3, SeqMatic, Fremont, Calif.). Following sequence assembly andannotation, the isolates containing nitrogen fixation gene clusters wereutilized in downstream research.

Microbiome Profiling of Isolation Seedlings

Genomic DNA was isolated from root-associated bacteria using the ZR-96Genomic DNA I Kit (Zymo Research P/N D3011), and 16S rRNA amplicons weregenerated using nextera-barcoded primers targeting 799f and 1114r. Theamplicon libraries were purified and sequenced with the Illumina MiSeqv3 platform (SeqMatic, Fremont, Calif.). Reads were taxonomicallyclassified using Kraken using the minikraken database (FIG. 2).

Acetylene Reduction Assay (ARA)

A modified version of the Acetylene Reduction Assay was used to measurenitrogenase activity in pure culture conditions. Strains were propagatedfrom single colony in SOB (RPI, P/N S25040-1000) at 30° C. with shakingat 200 RPM for 24 hours and then subcultured 1:25 into growth medium andgrown aerobically for 24 hours (30° C., 200 RPM). 1 ml of the minimalmedia culture was then added to 4 ml of minimal media supplemented with0 to 10 mM glutamine in air-tight Hungate tubes and grown anaerobicallyfor 4 hours (30° C., 200 RPM). 10% headspace was removed then replacedby an equal volume of acetylene by injection, and incubation continuedfor 1 hr. Subsequently, 2 ml of headspace was removed via gas tightsyringe for quantification of ethylene production using an Agilent 6850gas chromatograph equipped with a flame ionization detector (FID).

Ammonium Excretion Assay

Excretion of fixed nitrogen in the form of ammonia was measured usingbatch fermentation in anaerobic bioreactors. Strains were propagatedfrom single colony in 1 ml/well of SOB in a 96 well DeepWell plate. Theplate was incubated at 30° C. with shaking at 200 RPM for 24 hours andthen diluted 1:25 into a fresh plate containing 1 ml/well of growthmedium. Cells were incubated for 24 hours (30° C., 200 RPM) and thendiluted 1:10 into a fresh plate containing minimal medium. The plate wastransferred to an anaerobic chamber with a gas mixture of >98.5%nitrogen, 1.2-1.5% hydrogen and <30 ppM oxygen and incubated at 1350RPM, room temperature for 66-70 hrs. Initial culture biomass wascompared to ending biomass by measuring optical density at 590 nm. Cellswere then separated by centrifugation, and supernatant from the reactorbroth was assayed for free ammonia using the Megazyme Ammonia Assay kit(P/N K-AMIAR) normalized to biomass at each timepoint.

Extraction of Root-Associated Microbiome

Roots were shaken gently to remove loose particles, and root systemswere separated and soaked in a RNA stabilization solution (Thermo FisherP/N AM7021) for 30 minutes. The roots were then briefly rinsed withsterile deionized water. Samples were homogenized using bead beatingwith ½-inch stainless steel ball bearings in a tissue lyser (TissueLyserII, Qiagen P/N 85300) in 2 ml of lysis buffer (Qiagen P/N 79216).Genomic DNA extraction was performed with ZR-96 Quick-gDNA kit (ZymoResearch P/N D3010), and RNA extraction using the RNeasy kit (Qiagen P/N74104).

Root Colonization Assay

Four days after planting, 1 ml of a bacterial overnight culture(approximately 10⁹ cfu) was applied to the soil above the planted seed.Seedlings were fertilized three times weekly with 25 ml modifiedHoagland's solution supplemented with 0.5 mM ammonium nitrate. Fourweeks after planting, root samples were collected and the total genomicDNA (gDNA) was extracted. Root colonization was quantified using qPCRwith primers designed to amplify unique regions of either the wild typeor derivative strain genome. QPCR reaction efficiency was measured usinga standard curve generated from a known quantity of gDNA from the targetgenome. Data was normalized to genome copies per g fresh weight usingthe tissue weight and extraction volume. For each experiment, thecolonization numbers were compared to untreated control seedlings.

In Planta Transcriptomics

Transcriptional profiling of root-associated microbes was measured inseedlings grown and processed as described in the Root ColonizationAssay. Purified RNA was sequenced using the Illumina NextSeq platform(SeqMatic, Fremont, Calif.). Reads were mapped to the genome of theinoculated strain using bowtie2 using ‘—very-sensitive-local’ parametersand a minimum alignment score of 30. Coverage across the genome wascalculated using samtools. Differential coverage was normalized tohousekeeping gene expression and visualized across the genome usingCircos and across the nif gene cluster using DNAplotlib. Additionally,the in planta transcriptional profile was quantified via targetedNanostring analysis. Purified RNA was processed on an nCounter Sprint(Core Diagnostics, Hayward, Calif.).

15N Dilution Greenhouse Study

A 15N fertilizer dilution experiment was performed to assess optimizedstrain activity in planta. A planting medium containing minimalbackground N was prepared using a mixture of vermiculite and washed sand(5 rinses in DI H₂O). The sand mixture was autoclaved for 1 hour at 122°C. and approximately 600 g measured out into 40 cubic inch (656 mL)pots, which were saturated with sterile DI H₂O and allowed to drain 24hours before planting. Corn seeds (DKC 66-40) were surface sterilized in0.625% sodium hypochlorite for 10 minutes, then rinsed five times insterile distilled water and planted 1 cm deep. The plants weremaintained under fluorescent lamps for four weeks with 16-hour daylength at room temperatures averaging 22° C. (night) to 26° C. (day).

Five days after planting, seedlings were inoculated with a 1 mlsuspension of cells drenched directly over the emerging coleoptile.Inoculum was prepared from 5 ml overnight cultures in SOB, which werespun down and resuspended twice in 5 ml PBS to remove residual SOBbefore final dilution to OD of 1.0 (approximately 10⁹ CFU/ml). Controlplants were treated with sterile PBS, and each treatment was applied toten replicate plants.

Plants were fertilized with 25 ml fertilizer solution containing 2%15N-enriched 2 mM KNO₃ on 5, 9, 14, and 19 days after planting, and thesame solution without KNO₃ on 7, 12, 16, and 18 days after planting. Thefertilizer solution contained (per liter) 3 mmol CaCl₂, 0.5 mmol KH₂PO₄,2 mmol MgSO₄, 17.9 μmol FeSO₄, 2.86 mg H₃BO₃, 1.81 mg MnCl₂.4H₂O, 0.22mg ZnSO₄.7H₂O, 51 μg CuSO₄.5H₂O, 0.12 mg Na₂MoO₄.2H₂O, and 0.14 nmolNiCl₂. All pots were watered with sterile DI H₂O as needed to maintainconsistent soil moisture without runoff.

At four weeks, plants were harvested and separated at the lowest nodeinto samples for root gDNA and RNA extraction and aerial tissue forIRMS. Aerial tissues were wiped as needed to remove sand, placed wholeinto paper bags and dried for at least 72 hours at 60° C. Oncecompletely dry, total aerial tissue was homogenized by bead beating and5-7 mg samples were analyzed by isotope ratio mass spectrometry (IRMS)for δ15N by the MBL Stable Isotope Laboratory (The Ecosystems Center,Woods Hole, Mass.). Percent NDFA was calculated using the followingformula: % NDFA=(δ15N of UTC average−δ15N of sample)/(δ15N of UTCaverage)×100.

Example 3: Field Trials with Remodeled Microbes of the Disclosure—Summer2016

In order to evaluate the efficacy of remodeled strains of the presentdisclosure on corn growth and productivity under varying nitrogenregimes, field trials were conducted.

Trials were conducted with (1) seven subplot treatments of six strainsplus the control—four main plots comprised 0, 15, 85, and 100% ofmaximum return to nitrogen (MRTN) with local verification. The control(UTC only) was conducted with 10 100% MRTN plus, 5, 10, or 15 pounds.Treatments had four replications.

Plots of corn (minimum) were 4 rows of 30 feet in length, with 124 plotsper location. All observations were taken from the center two rows ofthe plots, and all destructive sampling was taken from the outside rows.Seed samples were refrigerated until 1.5 to 2 hours prior to use.

Local Agricultural Practice: The seed was a commercial corn withoutconventional fungicide and insecticide treatment. All seed treatmentswere applied by a single seed treatment specialist to assure uniformity.Planting date, seeding rate, weed/insect management, etc. were left tolocal agricultural practices. With the exception of fungicideapplications, standard management practices were followed.

Soil Characterization: Soil texture and soil fertility were evaluated.Soil samples were pre-planted for each replicate to insure residualnitrate levels lower than 50 lbs/Ac. Soil cores were taken from 0 cm to30 cm. The soil was further characterized for pH, CEC, total K and P.

Assessments: The initial plant population was assessed 14 days afterplanting (DAP)/acre, and were further assessed for: (1) vigor (1 to 10scale, w/10=excellent) 14 DAP & V10; (2) recordation of disease ratingsany time symptoms are evident in the plots; (3) record any differencesin lodging if lodging occurs in the plots; (4) yield (Bu/acre), adjustedto standard moisture pct; (5) test weight; and (6) grain moisturepercentage.

Sampling Requirements: The soil was sampled at three timepoints (priorto trial initiation, V10-VT, 1 week post-harvest). All six locations andall plots were sampled at 10 grams per sample (124 plots×3 timepoints×6locations).

Colonization Sampling: Colonization samples were collected at twotimepoints (V10 and VT) for five locations and six timepoints (V4, V8,V10, VT, R5, and Post-Harvest). Samples were collected as follows: (1)from 0% and 100% MRTN, 60 plots per location; (2) 4 plants per plotrandomly selected from the outside rows; (3) 5 grams of root, 8 inchesof stalk, and top three leaves—bagged and IDed each separately—12/bagsper plot; (4) five locations (60 plots×2 timepoints×12 bags/plot); andone location (60 plots×6 timepoints×12 bags/plot.

Normalized difference vegetation index (NDVI) determination was madeusing a Greenseeker instrument at two timepoints (V4-V6 and VT).Assessed each plot at all six locations (124 plots×2 timepoints×6locations).

Root analysis was performed with Win Rhizo from one location that bestillustrated treatment differentiation. Ten plants per plot were randomlysampled (5 adjacent from each outside row; V3-V4 stage plants werepreferred) and gently washed to remove as much dirt as reasonable. Tenroots were placed in a plastic bag and labelled. Analyzed with WinRhizoRoot Analysis.

Stalk Characteristics were measured at all six locations between R2 andR5. The stalk diameter of ten plants per plot at the 6″ height wererecorded, as was the length of the first internode above the 6″ mark.Ten plants were monitored; five consecutive plants from the center ofthe two inside rows. Six locations were evaluated (124 plots×2measures×6 locations).

The tissue nitrates were analyzed from all plots and all locations. An8″ segment of stalk beginning 6″ above the soil when the corn is betweenone and three weeks after black layer formation; leaf sheaths wereremoved. All locations and plots were evaluated (6 locations×124 plots).

The following weather data was recorded for all locations from plantingto harvest: daily maximum and minimum temperatures, soil temperature atseeding, daily rainfall plus irrigation (if applied), and any unusualweather events such as excessive rain, wind, cold, or heat.

Yield data across all six locations is presented in Table 26. Nitrogenrate had a significant impact on yield, but strains across nitrogenrates did not. However, at the lowest nitrogen rate, strains C1006,CM029, and C1019 numerically out-yielded the UTC by 4 to 6 bu/acre.Yield was also numerically increased 2 to 4 bu/acre by strains CM029,C1019, and CM081 at 15% MRTN.

TABLE 26 Yield data across all six locations Stalk Internode YLD (bu)Vigor_E Vigor_L Diameter (mm) Length (in) NDVI_Veg NDVI_Rep MRTN % 0143.9 7.0 5.7 18.87 7.18 64.0 70.6 15 165.9 7.2 6.3 19.27 7.28 65.8 72.585 196.6 7.1 7.1 20.00 7.31 67.1 74.3 100 197.3 7.2 7.2 20.23 7.37 66.372.4 Strain CI006 (1) 176.6 7.2 6.6 19.56 18.78 66.1 72.3 CM029 (2)176.5 7.1 6.5 19.54 18.61 65.4 71.9 CM038 (3) 175.5 7.2 6.5 19.58 18.6965.7 72.8 CI019 (4) 176.0 7.1 6.6 19.51 18.69 65.5 72.9 CM081 (5) 176.27.1 6.6 19.57 18.69 65.8 73.1 CM029/CM081 (6) 174.3 7.1 6.6 19.83 18.7966.2 72.5 UTC (7) 176.4 7.1 6.6 19.54 18.71 65.9 71.7 MRTN/Strain 0 1145.6 7.0 5.6 19.07 7.12 63.5 70.3 0 2 147.0 7.0 5.5 18.74 7.16 64.470.4 0 3 143.9 7.0 5.5 18.83 7.37 64.6 70.5 0 4 146.0 6.9 5.7 18.86 7.1563.4 70.7 0 5 141.7 7.0 5.8 18.82 7.05 63.6 70.9 0 6 142.2 7.2 5.8 19.127.09 64.7 69.9 0 7 141.2 7.0 5.8 18.64 7.32 64.0 71.4 15 1 164.2 7.3 6.119.09 7.21 66.1 71.5 15 2 167.3 7.2 6.3 19.32 7.29 65.5 72.7 15 3 165.67.3 6.3 19.36 7.23 64.8 72.5 15 4 167.9 7.3 6.4 19.31 7.51 66.1 72.3 155 169.3 7.2 6.2 19.05 7.32 66.0 72.8 15 6 161.9 7.1 6.3 19.45 7.20 66.272.2 15 7 165.1 7.3 6.4 19.30 7.18 66.0 73.3 85 1 199.4 7.3 7.2 19.707.32 67.2 74.0 85 2 195.1 7.1 7.2 19.99 7.09 66.5 74.4 85 3 195.0 7.07.0 20.05 7.26 67.3 74.6 85 4 195.6 7.2 7.1 20.04 7.29 66.4 74.4 85 5196.4 7.2 7.0 19.87 7.39 67.3 74.5 85 6 195.1 7.0 6.9 20.35 7.34 67.474.4 85 7 199.5 6.9 7.2 19.97 7.48 67.4 74.1 100 1 197.1 7.2 7.3 20.387.68 67.5 73.4 100 2 196.5 7.0 7.1 20.11 7.21 65.3 70.2 100 3 197.6 7.57.3 20.08 7.42 66.3 73.4 100 4 194.6 7.1 7.1 19.83 7.40 66.1 74.1 100 5197.4 7.2 7.3 20.53 7.36 66.2 74.3 100 6 198.1 7.2 7.4 20.40 7.16 66.673.6 100 7 199.9 7.2 7.2 20.26 7.32 66.2 68.1

Another analysis approach is presented in Table 27. The table comprisesthe four locations where the response to nitrogen was the greatest whichsuggests that available residual nitrogen was lowest. This approach doesnot alter the assessment that the nitrogen rate significantly impactedyield, which strains did not when averaged across all nitrogen rates.However, the numerical yield advantage at the lowest N rate is morepronounced for all strains, particularly C1006, CM029, and CM029/CM081where yields were increased from 8 to 10 bu/acre. At 15% MRTN, strainCM081 outyielded UTC by 5 bu.

TABLE 27 Yield data across four locations 4 Location Average - SGS,Agldea, Bennett, RFR Stalk Diameter Internode YLD (bu) Vigor_E Vigor_L(mm) Length (in) MRTN %  0 137.8 7.3 5.84 18.10 5.36  15 162.1 7.5 6.6318.75 5.40  85 199.2 7.4 7.93 19.58 5.62 100 203.5 7.5 8.14 19.83 5.65Strain CI006 (1) 175.4 7.5 7.08 19.03 5.59 CM029 (2) 176.1 7.4 7.0819.09 5.39 CM038 (3) 175.3 7.5 7.05 19.01 5.59 CI019 (4) 174.8 7.5 7.1619.02 5.45 CM081 (5) 176.7 7.4 7.16 19.00 5.53 CM029/CM081 (6) 175.1 7.47.17 19.33 5.46 UTC (7) 176.0 7.3 7.27 18.98 5.55 MRTN/Strain 0 1 140.07.3 5.69 18.32 5.28 0 2 140.7 7.4 5.69 18.19 5.23 0 3 135.5 7.3 5.6317.95 5.50 0 4 138.8 7.3 5.81 17.99 5.36 0 5 136.3 7.3 6.06 18.05 5.34 06 141.4 7.5 6.00 18.43 5.30 0 7 131.9 7.3 6.00 17.75 5.48 15 1 158.0 7.66.44 18.53 5.34 15 2 164.1 7.5 6.56 19.13 5.42 15 3 164.3 7.6 6.63 18.685.51 15 4 163.5 7.6 6.81 18.84 5.34 15 5 166.8 7.5 6.63 18.60 5.39 15 6156.6 7.4 6.56 18.86 5.41 15 7 161.3 7.5 6.81 18.62 5.42 85 1 199.4 7.68.00 19.15 5.63 85 2 199.0 7.4 8.09 19.49 5.46 85 3 198.2 7.4 7.75 19.885.69 85 4 196.8 7.4 8.00 19.65 5.60 85 5 199.5 7.4 7.75 19.26 5.70 85 6198.7 7.3 7.81 19.99 5.61 85 7 202.8 7.2 8.13 19.66 5.65 100 1 204.3 7.48.19 20.11 6.10 100 2 200.6 7.3 8.00 19.53 5.46 100 3 203.3 7.7 8.1919.55 5.67 100 4 200.2 7.6 8.00 19.59 5.49 100 5 203.9 7.4 8.19 20.085.68 100 6 203.8 7.5 8.31 20.05 5.52 100 7 208.1 7.4 8.13 19.90 5.63

The results from the field trial are also illustrated in FIGS. 9-15. Theresults indicate that the microbes of the disclosure are able toincrease plant yield, which points to the ability of the taught microbesto increase nitrogen fixation in an important agricultural crop, i.e.corn.

The field based results further validate the disclosed methods ofnon-intergenerically modifying the genome of selected microbial strains,in order to bring about agriculturally relevant results in a fieldsetting when applying said engineered strains to a crop.

FIG. 6 depicts the lineage of modified remodeled strains that werederived from strain C1006 (WT Kosakonia sacchari). The field datademonstrates that an engineered derivative of the C1006 WT strain, i.e.CM029, is able to bring about numerically relevant results in a fieldsetting. For example, Table 26 illustrates that at 0% MRTN CM029 yielded147.0 bu/acre compared to untreated control at 141.2 bu/acre (anincrease of 5.8 bu/acre). Table 26 also illustrates that at 15% MRTNCM029 yielded 167.3 bu/acre compared to untreated control at 165.1bu/acre (an increase of 2.2 bu/acre). Table 27 is supportive of theseconclusions and illustrates that at 0% MRTN CM029 yielded 140.7 bu/acrecompared to untreated control at 131.9 bu/acre (an increase of 8.8bu/acre). Table 27 also illustrates that at 15% MRTN CM029 yielded 164.1bu/acre compared to untreated control at 161.3 bu/acre (an increase of2.8 bu/acre).

FIG. 7 depicts the lineage of modified remodeled strains that werederived from strain C1019 (WT Rahnella aquatilis). The field datademonstrates that an engineered derivative of the C1019 WT strain, i.e.CM081, is able to bring about numerically relevant results in a fieldsetting. For example, Table 26 illustrates that at 15% MRTN CM081yielded 169.3 bu/acre compared to untreated control at 165.1 bu/acre (anincrease of 4.2 bu/acre). Table 27 is supportive of these conclusionsand illustrates that at 0% MRTN CM081 yielded 136.3 bu/acre compared tountreated control at 131.9 bu/acre (an increase of 4.4 bu/acre). Table27 also illustrates that at 15% MRTN CM081 yielded 166.8 bu/acrecompared to untreated control at 161.3 bu/acre (an increase of 5.5bu/acre).

Further, one can see in Table 27 that the combination of CM029/CM081 at0% MRTN yielded 141.4 bu/acre compared to untreated control at 131.9bu/acre (an increase of 9.5 bu/acre).

Example 4: Field Trials with Remodeled Microbes of the Disclosure—Summer2017

In order to evaluate the efficacy of remodeled strains of the presentdisclosure on corn growth and productivity under varying nitrogenregimes, field trials were conducted. The below field data demonstratesthat the non-intergeneric microbes of the disclosure are able to fixatmospheric nitrogen and deliver said nitrogen to a plant—resulting inincreased yields—in both a nitrogen limiting environment, as well as anon-nitrogen limiting environment.

Trials were conducted at seven locations across the United States withsix geographically diverse Midwestern locations. Five nitrogen regimeswere used for fertilizer treatments: 100% of standard agriculturalpractice of the site/region, 100% minus 25 pounds, 100% minus 50 pounds,100% minus 75 pounds, and 0%; all per acre. The pounds of nitrogen peracre for the 100% regime depended upon the standard agriculturalpractices of the site/region. The aforementioned nitrogen regimes rangedfrom about 153 pounds per acre to about 180 pounds per acre, with anaverage of about 164 pounds of nitrogen per acre.

Within each fertilizer regime, there were 14 treatments. Each regime hadsix replications, and a split plot design was utilized. The 14treatments included: 12 different microbes, 1 UTC with the samefertilizer rate as the main plot, and 1 UTC with 100% nitrogen. In the100% nitrogen regime the 2nd UTC is 100 plus 25 pounds.

Plots of corn, at a minimum, were 4 rows of 30 feet in length (30 inchesbetween rows) with 420 plots per location. All observations, unlessotherwise noted, were taken from the center two rows of the plants, andall destructive sampling was taken from the outside rows. Seed sampleswere refrigerated until 1.5 to 2 hours prior to use.

Local Agricultural Practice: The seed was a commercial corn applied witha commercial seed treatment with no biological co-application. Theseeding rate, planting date, weed/insect management, harvest times, andother standard management practices were left to the norms of localagricultural practices for the regions, with the exception of fungicideapplication (if required).

Microbe Application: The microbes were applied to the seed in a seedtreatment over seeds that had already received a normal chemicaltreatment. The seed were coated with fermentation broth comprising themicrobes.

Soil Characterization: Soil texture and soil fertility were evaluated.Standard soil sampling procedures were utilized, which included soilcores of depths from 0-30 cm and 30-60 cm. The standard soil samplingincluded a determination of nitrate nitrogen, ammonium nitrogen, totalnitrogen, organic matter, and CEC. Standard soil sampling furtherincluded a determination of pH, total potassium, and total phosphorous.To determine the nitrogen fertilizer levels, preplant soil samples fromeach location were taken to ensure that the 0-12″ and potentially the12″ to 24″ soil regions for nitrate nitrogen.

Prior to planting and fertilization, 2 ml soil samples were collectedfrom 0 to 6-12″ from the UTC. One sample per replicate per nitrogenregion was collected using the middle of the row. (5 fertilizerregimes×6 replicates=thirty soil samples).

Post-planting (V4-V6), 2 ml soil samples were collected from 0 to 6-12″from the UTC. One sample per replicate per nitrogen region was collectedusing the middle of the row. (5 fertilizer regimes×6 replicates=thirtysoil samples).

Post-harvest (V4-V6), 2 ml soil samples were collected from 0 to 6-12″from the UTC. One sample per replicate per nitrogen region was collectedusing the middle of the row. Additional post-harvest soil samplecollected at 0-12″ from the UTC and potentially 12-24″ from the UTC (5fertilizer regimes×6 replicates=thirty soil samples).

A V6-V10 soil sample from each fertilizer regime (excluding thetreatment of 100% and 100%+25 lbs [in the 100% block] for all fertilizerregimes at 0-12″ and 12-24″. (5 fertilizer regimes×2 depths=10 samplesper location).

Post-harvest soil sample from each fertilizer regime (excluding thetreatment of 100% and 100%+25 lbs [in the 100% block] for all fertilizerregimes at 0-12″ and 12-24″. (5 fertilizer regimes×2 depths=10 samplesper location).

Assessments: The initial plant population was assessed at ˜50% UTC andthe final plant population was assessed prior to harvest. Assessmentincluded (1) potentially temperature (temperature probe); (2) vigor(1-10 scale with 10=excellent) at V4 and V8-V10; (3) plant height atV8-V10 and V14; (4) yield (bushels/acre) adjusted to standard moisturepercentage; (5) test weight; (6) grain moisture percentage; (7) stalknitrate tests at black layer (420 plots×7 locations); (8) colonizationwith 1 plant per plot in zip lock bag at 0% and 100% fertilizer at V4-V6(1 plant×14 treatments×6 replicates×2 fertilizer regimes=168 plants);(9) transcriptomics with 1 plant per plot in zip lock bag at 0% and 100%fertilizer at V4-V6 (1 plant×14 treatments×6 replicates×2 fertilizerregimes=168 plants); (10) Normalized difference vegetative index (NDVI)or normalized difference red edge (NDRE) determination using aGreenseeker instrument at two time points (V4-V6 and VT) to assess eachplot at all 7 locations (420 plots×2 time points×7 locations=5,880 datapoints); (11) stalk characteristics measured at all 7 locations betweenR2 and R5 by recording the stalk diameter of 10 plants/plot at 6″height, record length of first internode above the 6″ mark, 10 plantsmonitored (5 consecutive plants from center of two inside rows) (420plots×10 plants×7 locations=29,400 data points).

Monitoring Schedule: Practitioners visited all trials at V3-V4 stage toassess early-season response to treatments and during reproductivegrowth stage to monitor maturity. Local cooperator visited researchtrial on an on-going basis.

Weather Information: Weather data spanning from planting to harvest wascollected and consisted of daily minimum and maximum temperatures, soiltemperature at seeding, daily rainfall plus irrigation (if applied), andunusual weather events such as excessive wind, rain, cold, heat.

Data Reporting: Including the data indicated above, the field trialsgenerated data points including soil textures; row spacing; plot sizes;irrigation; tillage; previous crop; seeding rate; plant population;seasonal fertilizer inputs including source, rate, timing, andplacement; harvest area dimensions, method of harvest, such as by handor machine and measurement tools used (scales, yield monitor, etc.)

Results: Select results from the aforementioned field trial are reportedin FIG. 16 and FIG. 17.

In FIG. 16, it can be seen that a remodeled microbe of the disclosure(i.e. 6-403) resulted in a higher yield than the wild type strain (WT)and a higher yield than the untreated control (UTC). The “−25 lbs N”treatment utilizes 25 lbs less N per acre than standard agriculturalpractices of the region. The “100% N” UTC treatment is meant to depictstandard agricultural practices of the region, in which 100% of thestandard utilization of N is deployed by the farmer. The microbe “6-403”was deposited as NCMA 201708004 and can be found in Table 1. This is amutant Kosakonia sacchari (also called CM037) and is a progeny mutantstrain from CI006 WT.

In FIG. 17, the yield results obtained demonstrate that the remodeledmicrobes of the disclosure perform consistently across locations.Furthermore, the yield results demonstrate that the microbes of thedisclosure perform well in both a nitrogen stressed environment (i.e. anitrogen limiting environment), as well as an environment that hassufficient supplies of nitrogen (i.e. a non-nitrogen-limitingcondition). The microbe “6-881” (also known as CM094, PBC6.94), andwhich is a progeny mutant Kosakonia sacchari strain from CI006 WT, wasdeposited as NCMA 201708002 and can be found in Table 1. The microbe“137-1034,” which is a progeny mutant Klebsiella variicola strain fromCI137 WT, was deposited as NCMA 201712001 and can be found in Table 1.The microbe “137-1036,” which is a progeny mutant Klebsiella variicolastrain from CI137 WT, was deposited as NCMA 201712002 and can be foundin Table 1. The microbe “6-404” (also known as CM38, PBC6.38), and whichis a progeny mutant Kosakonia sacchari strain from CI006 WT, wasdeposited as NCMA 201708003 and can be found in Table 1.

Example 5: Genus of Non-Intergeneric Remodeled Microbes Beneficial forAgricultural Systems

The remodeled microbes of the present disclosure were evaluated andcompared against one another for the production of nitrogen produced inan acre across a season. See FIG. 8, FIG. 24, and FIG. 25.

It is hypothesized by the inventors that in order for a population ofengineered non-intergeneric microbes to be beneficial in a modern rowcrop agricultural system, then the population of microbes needs toproduce at least one pound or more of nitrogen per acre per season.

To that end, the inventors have surprisingly discovered a functionalgenus of microbes that are able to contribute, inter alia, to:increasing yields in non-leguminous crops; and/or lessening a farmer'sdependence upon exogenous nitrogen application; and/or the ability toproduce at least one pound of nitrogen per acre per season, even innon-nitrogen-limiting environments, said genus being defined by theproduct of colonization ability×mmol of N produced per microbe per hour(i.e. the line partitioning FIGS. 8, 24, and 25).

With respect to FIGS. 8, 24, and 25, certain data utilizing microbes ofthe disclosure was aggregated, in order to depict a heatmap of thepounds of nitrogen delivered per acre-season by microbes of thedisclosure, which are recorded as a function of microbes per g-freshweight by mmol of nitrogen/microbe-hr. Below the thin line thattransects the larger images are the microbes that deliver less than onepound of nitrogen per acre-season, and above the line are the microbesthat deliver greater than one pound of nitrogen per acre-season.

Field Data & Wild Type Colonization Heatmap: The microbes utilized inthe FIG. 8 heatmap were assayed for N production in corn. For the WTstrains CI006 and CI019, corn root colonization data was taken from asingle field site. For the remaining strains, colonization was assumedto be the same as the WT field level. N-fixation activity was determinedusing an in vitro ARA assay at 5 mM glutamine. The table below theheatmap in FIG. 8 gives the precise value of mmol N produced per microbeper hour (mmol N/Microbe hr) along with the precise CFU per gram offresh weight (CFU/g fw) for each microbe shown in the heatmap.

Field Data Heatmap: The data utilized in the FIG. 24 heatmap is derivedfrom microbial strains assayed for N production in corn in fieldconditions. Each point represents lb N/acre produced by a microbe usingcorn root colonization data from a single field site. N-fixationactivity was determined using in vitro ARA assay at 5 mM N in the formof glutamine or ammonium phosphate. The below Table 28 gives the precisevalue of mmol N produced per microbe per hour (mmol N/Microbe hr) alongwith the precise CFU per gram of fresh weight (CFU/g fw) for eachmicrobe shown in the heatmap of FIG. 24.

Greenhouse & Laboratory Data Heatmap: The data utilized in the FIG. 25heatmap is derived from microbial strains assayed for N production incorn in laboratory and greenhouse conditions. Each point represents lbN/acre produced by a single strain. White points represent strains inwhich corn root colonization data was gathered in greenhouse conditions.Black points represent mutant strains for which corn root colonizationlevels are derived from average field corn root colonization levels ofthe wild-type parent strain. Hatched points represent the wild typeparent strains at their average field corn root colonization levels. Inall cases, N-fixation activity was determined by in vitro ARA assay at 5mM N in the form of glutamine or ammonium phosphate. The below Table 29gives the precise value of mmol N produced per microbe per hour (mmolN/Microbe hr) along with the precise CFU per gram of fresh weight (CFU/gfw) for each microbe shown in the heatmap of FIG. 25.

TABLE 28 FIG. 24 - Field Data Heatmap Activity Peak Strain (mmol N/Colonization N Produced/ Name Microbe hr) (CFU/g fw) acre seasonTaxonomic Designation CI006 3.88E−16 1.50E+07 0.24 Kosakonia sacchari 6-404 1.61E−13 3.50E+05 2.28 Kosakonia sacchari  6-848 1.80E−132.70E+05 1.97 Kosakonia sacchari  6-881 1.58E−13 5.00E+05 3.20 Kosakoniasacchari  6-412 4.80E−14 1.30E+06 2.53 Kosakonia sacchari  6-4031.90E−13 1.30E+06 10.00 Kosakonia sacchari CI019 5.33E−17 2.40E+06 0.01Rahnella aquatilis 19-806 6.65E−14 2.90E+06 7.80 Rahnella aquatilis19-750 8.90E−14 2.60E+05 0.94 Rahnella aquatilis 19-804 1.72E−144.10E+05 0.29 Rahnella aquatilis CI137 3.24E−15 6.50E+06 0.85 Klebsiellavariicola 137-1034 1.16E−14 6.30E+06 2.96 Klebsiella variicola 137-10363.47E−13 1.30E+07 182.56 Klebsiella variicola 137-1314 1.70E−13 1.99E+040.14 Klebsiella variicola 137-1329 1.65E−13 7.25E+04 0.48 Klebsiellavariicola 63 3.60E−17 3.11E+05 0.00 Rahnella aquatilis  63-1146 1.90E−145.10E+05 0.39 Rahnella aquatilis 1021  1.77E−14 2.69E+07 19.25 Kosakoniapseudosacchari 728  5.56E−14 1445240.09 3.25 Klebsiella variicola

TABLE 29 FIG. 25 - Greenhouse & Laboratory Data Heatmap Activity PeakStrain (mmol N/ Colonization N Produced/ Name Microbe hr) (CFU/g fw)acre season Taxonomic Designation CI006 3.88E−16 1.50E+07 0.24 Kosakoniasacchari  6-400 2.72E−13 1.79E+05 1.97 Kosakonia sacchari  6-3971.14E−14 1.79E+05 0.08 Kosakonia sacchari CI137 3.24E−15 6.50E+06 0.85Klebsiella variicola  137-1586 1.10E−13 1.82E+06 8.10 Klebsiellavariicola  137-1382 4.81E−12 1.82E+06 354.60 Klebsiella variicola 1021 1.77E−14 2.69E+07 19.25 Kosakonia pseudosacchari 1021-1615 1.20E−132.69E+07 130.75 Kosakonia pseudosacchari 1021-1619 3.93E−14 2.69E+0742.86 Kosakonia pseudosacchari 1021-1612 1.20E−13 2.69E+07 130.75Kosakonia pseudosacchari 1021-1623 4.73E−17 2.69E+07 0.05 Kosakoniapseudosacchari 1293  5.44E−17 8.70E+08 1.92 Azospirillum lipoferum 1116 1.05E−14 1.37E+07 5.79 Enterobacter sp. 1113  8.05E−15 4.13E+07 13.45Enterobacter sp. 910 1.19E−13 1.34E+06 6.46 Kluyvera intermedia 910-1246 2.16E−13 1.34E+06 11.69 Kluyvera intermedia 850 7.2301E−16 1.17E+06 0.03 Achromobacter spiritinus 852 5.96E−16 1.07E+06 0.03Achromobacter marplatensis 853 6.42E−16 2.55E+06 0.07 Microbacteriummurale

Conclusions: The data in FIGS. 8, 24, 25, and Tables 28 and 29,illustrates more than a dozen representative members of the describedgenus (i.e. microbes to the right of the line in the figures). Further,these numerous representative members come from a diverse array oftaxonomic genera, which can be found in the above Tables 28 and 29.Further still, the inventors have discovered numerous genetic attributesthat depict a structure/function relationship that is found in many ofthe microbes. These genetic relationships can be found in the numeroustables of the disclosure setting forth the genetic modificationsintroduced by the inventors, which include introducing at least onegenetic variation into at least one gene, or non-coding polynucleotide,of the nitrogen fixation or assimilation genetic regulatory network.

Consequently, the newly discovered genus is supported by: (1) a robustdataset, (2) over a dozen representative members, (3) members fromdiverse taxonomic genera, and (4) classes of genetic modifications thatdefine a structure/function relationship, in the underlying geneticarchitecture of the genus members.

Example 6: Methods and Assays for Detection of Non-IntergenericRemodeled Microbes

The present disclosure teaches primers, probes, and assays that areuseful for detecting the microbes utilized in the various aforementionedExamples. The assays are able to detect the non-natural nucleotide“junction” sequences in the derived/mutant non-intergeneric remodeledmicrobes. These non-naturally occurring nucleotide junctions can be usedas a type of diagnostic that is indicative of the presence of aparticular genetic alteration in a microbe.

The present techniques are able to detect these non-naturally occurringnucleotide junctions via the utilization of specialized quantitative PCRmethods, including uniquely designed primers and probes. The probes canbind to the non-naturally occurring nucleotide junction sequences. Thatis, sequence-specific DNA probes consisting of oligonucleotides that arelabelled with a fluorescent reporter, which permits detection only afterhybridization of the probe with its complementary sequence can be used.The quantitative methods can ensure that only the non-naturallyoccurring nucleotide junction will be amplified via the taught primers,and consequently can be detected via either a non-specific dye, or viathe utilization of a specific hybridization probe. Another aspect of themethod is to choose primers such that the primers flank either side of ajunction sequence, such that if an amplification reaction occurs, thensaid junction sequence is present.

Consequently, genomic DNA can be extracted from samples and used toquantify the presence of microbes of the disclosure by using qPCR. Theprimers utilized in the qPCR reaction can be primers designed by PrimerBlast (www.ncbi.nlm.nih.gov/tools/primer-blast/) to amplify uniqueregions of the wild-type genome or unique regions of the engineerednon-intergeneric mutant strains. The qPCR reaction can be carried outusing the SYBR GreenER qPCR SuperMix Universal (Thermo Fisher P/N11762100) kit, using only forward and reverse amplification primers;alternatively, the Kapa Probe Force kit (Kapa Biosystems P/N KK4301) canbe used with amplification primers and a TaqMan probe containing a FAMdye label at the 5′ end, an internal ZEN quencher, and a minor groovebinder and fluorescent quencher at the 3′ end (Integrated DNATechnologies).

Certain primer, probe, and non-native junction sequences—which can beused in the qPCR methods—are listed in the below Table 30. Specifically,the non-native junction sequences can be found in SEQ ID NOs: 372-405and 425-430.

TABLE 30 Microbial Detection up/ down 100 bp Junction  Junc- stream SEQ100 bp SEQ downstream SEQ SEQ “/” F R base tion junc- ID upstream of IDof ID indicating Junction primer primer Probe CI Name tion NO junctionNO junction NO junction des. SEQ SEQ SEQ 1021 ds1131 up 304 TGGTGTCCGGGC338 TTCTTGGTTCTCT 372 5′- disrupted N/A N/A N/A GAACGTCGCCAGGGAGCGCTTTAT TGGTGTCCGGGC nifL gene/ GTGGCACAAATT CGGCATCCTGACGAACGTCGCCAG PinfC GTCAGAACTACG TGAAGAATTTGC GTGGCACAAATT ACACGACTAACCAGGCTTCTTCCCA GTCAGAACTACG GACCGCAGGAGT ACCTGGCTTGCA ACACGACTAACCGTGCGATGACCC CCCGTGCAGGTA GACCGCAGGAGT TGAATATGATGA GTTGTGATGAACGTGCGATGACCC TGGA AT TGAATATGATGA TGGA/ TTCTTGGTTCTC TGGAGCGCTTTATCGGCATCCTGA CTGAAGAATTTG CAGGCTTCTTCC CAACCTGGCTTG CACCCGTGCAGGTAGTTGTGATGA ACAT-3′ 1021 ds1131 down 305 CGGAAAACGAGT 339 GCGATAGAACTC373 5′- PinfC/ N/A N/A N/A TCAAACGGCGCG ACTTCACGCCCC CGGAAAACGAGTdisrupted TCCCAATCGTATT GAAGGGGGAAGC TCAAACGGCGCG nifL gene AATGGCGAGATTTGCCTGACCCTAC TCCCAATCGTAT CGCGCCACGGAA GATTCCCGCTATT TAATGGCGAGATGTTCGCTTAACAG TCATTCACTGACC TCGCGCCACGGA GTCTGGAAGGCG GGAGGTTCAAAAAGTTCGCTTAAC AGCAGCTTGGTA TGACCCAGCGAA AGGTCTGGAAGG TT C CGAGCAGCTTGGTATT/ GCGATAGAACTC ACTTCACGCCCC GAAGGGGGAAGC TGCCTGACCCTA CGATTCCCGCTATTTCATTCACTG ACCGGAGGTTCA AAATGACCCAGC GAAC-3′ 1021 ds1133 N/A 306CGCCAGAGAGTT 340 TCCCTGTGCGCCG 374 5′- 5′ UTR N/A N/A N/A GAAATCGAACATCGTCGCCGATGG CGCCAGAGAGTT and ATG/ TTCCGTAATACCG TGGCCAGCCAACGAAATCGAACAT truncated CCATTACCCAGG TGGCGCGCTACC TTCCGTAATACC glnE geneAGCCGTTCTGGTT CGATCCTGCTCG GCCATTACCCAG GCACAGCGGAAA ATGAACTGCTCGGAGCCGTTCTGG ACGTTAACGAAA ACCCGAACACGC TTGCACAGCGGA GGATATTTCGCATTCTATCAACCGA AAACGTTAACGA G CGG AAGGATATTTCG CATG/ TCCCTGTGCGCCGCGTCGCCGATG GTGGCCAGCCAA CTGGCGCGCTAC CCGATCCTGCTC GATGAACTGCTCGACCCGAACACG CTCTATCAACCG ACGG-3′ 1021 ds1145 up 307 CGGGCGAACGTC 341CGTTCTGTAATAA 375 5′- disrupted N/A N/A N/A GCCAGGTGGCAC TAACCGGACAATCGGGCGAACGTC nifL gene/ AAATTGTCAGAA TCGGACTGATTA GCCAGGTGGCAC Prm1CTACGACACGAC AAAAAGCGCCCT AAATTGTCAGAA TAACCGACCGCA CGCGGCGCTTTTTCTACGACACGAC GGAGTGTGCGAT TTATATTCTCGAC TAACCGACCGCA GACCCTGAATATTCCATTTAAAATA GGAGTGTGCGAT GATGATGGATGC AAAAATCCAATC GACCCTGAATAT CAGCGATGATGGATGC CAGC/ CGTTCTGTAATA ATAACCGGACAA TTCGGACTGATT AAAAAAGCGCCCTCGCGGCGCTTT TTTTATATTCTC GACTCCATTTAA AATAAAAAATCC AATC-3′ 1021 ds1145down 308 TCAACCTAAAAA 342 AACTCACTTCAC 376 5′- Prm1/ N/A N/A N/AAGTTTGTGTAATA GCCCCGAAGGGG TCAACCTAAAAA disrupted CTTGTAACGCTACGAAGCTGCCTGA AGTTTGTGTAAT nifL gene ATGGAGATTAAC CCCTACGATTCCCACTTGTAACGCT TCAATCTAGAGG GCTATTTCATTCA ACATGGAGATTA GTATTAATAATGCTGACCGGAGGT ACTCAATCTAGA AATCGTACTAAA TCAAAATGACCC GGGTATTAATAACTGGTACTGGGC AGCGAACCGAGT TGAATCGTACTA GC CG AACTGGTACTGG GCGC/AACTCACTTCAC GCCCCGAAGGGG GAAGCTGCCTGA CCCTACGATTCC CGCTATTTCATTCACTGACCGGAG GTTCAAAATGAC CCAGCGAACCGA GTCG-3′ 1021 ds1148 up 309CGGGCGAACGTC 343 CGCGTCAGGTTG 377 5′- disrupted N/A N/A N/A GCCAGGTGGCACAACGTAAAAAAG CGGGCGAACGTC nifL gene/ AAATTGTCAGAA TCGGTCTGCGCAGCCAGGTGGCAC Prm7 CTACGACACGAC AAGCACGTCGTC AAATTGTCAGAA TAACCGACCGCAGTCCGCAGTTCTC CTACGACACGAC GGAGTGTGCGAT CAAACGTTAATT TAACCGACCGCAGACCCTGAATAT GGTTTCTGCTTCG GGAGTGTGCGAT GATGATGGATGC GCAGAACGATTGGACCCTGAATAT CAGC GC GATGATGGATGC CAGC/ CGCGTCAGGTTG AACGTAAAAAAGTCGGTCTGCGCA AAGCACGTCGTC GTCCGCAGTTCT CCAAACGTTAAT TGGTTTCTGCTTCGGCAGAACGAT TGGC-3′ 1021 ds1148 down 310 AATTTTCTGCCCA 344 AACTCACTTCAC378 5′- Prm4/ N/A N/A N/A AATGGCTGGGAT GCCCCGAAGGGG AATTTTCTGCCCdisrupted TGTTCATTTTTTG GAAGCTGCCTGA AAATGGCTGGGA nifL geneTTTGCCTTACAAC CCCTACGATTCCC TTGTTCATTTTT GAGAGTGACAGT GCTATTTCATTCATGTTTGCCTTAC ACGCGCGGGTAG CTGACCGGAGGT AACGAGAGTGAC TTAACTCAACATCTCAAAATGACCC AGTACGCGCGGG TGACCGGTCGAT AGCGAACCGAGT TAGTTAACTCAA CGCATCTGACCGGT CGAT/ AACTCACTTCAC GCCCCGAAGGGG GAAGCTGCCTGA CCCTACGATTCCCGCTATTTCATT CACTGACCGGAG GTTCAAAATGAC CCAGCGAACCGA GTCG-3′ CI006 ds126N/A 311 GTAACCAATAAA 345 CCGATCCCCATC 379 5′- 5′ UTR up N/A N/A N/AGGCCACCACGCC ACTGTGTGTCTTG GTAACCAATAAA to ATG- AGACCACACGATTATTACAGTGCC GGCCACCACGCC 4 bp of AGTGATGGCAAC GCTTCGTCGGCTTAGACCACACGAT amtB gene/ ACTTTCCAGCTGC CGCCGGTACGAA AGTGATGGCAACdisrupted ACCAGCACCTGA TACGAATGACGC ACTTTCCAGCTG amtB gene TGGCCCATGGTCGTTGCAGCTCAG CACCAGCACCTG ACACCTTCAGCG CAACGAAAATTT ATGGCCCATGGT AAA TGCACACCTTCAGC GAAA/ CCGATCCCCATC ACTGTGTGTCTT GTATTACAGTGC CGCTTCGTCGGCTTCGCCGGTACG AATACGAATGAC GCGTTGCAGCTC AGCAACGAAAAT TTTG-3′ CI019 ds172down 312 TGGTATTGTCAGT 346 CCGTCTCTGAAG 380 5′- Prm1.2/ SEQ SEQ N/ACTGAATGAAGCT CTCTCGGTGAAC TGGTATTGTCAG disrupted ID ID CTTGAAAAAGCTATTGTTGCGAGG TCTGAATGAAGC nifL gene NO: NO: GAGGAAGCGGGC CAGGATGCGAGCTCTTGAAAAAGC 406 407 GTCGATTTAGTAG TGGTTGTGTTTTG TGAGGAAGCGGG CAAG TGCCAAATCAGTCCGA ACATTACCGATA CGTCGATTTAGT AAGT TCGC ATGCCGAGCCGCATGTGCCGCGTG AGAAATCAGTCC TCGC AACA CAGTTTGTCGAAT AACGGGTGCGTTGAATGCCGAGCC CTCA ATGT C ATG GCCAGTTTGTCG CAGG TCAC AATC/ CCGTCTCTGAAGCTCTCGGTGAAC ATTGTTGCGAGG CAGGATGCGAGC TGGTTGTGTTTT GACATTACCGATAATGTGCCGCGT GAACGGGTGCGT TATG-3′ CI019 ds172 up 313 ACCGATCCGCAG 347TGAACATCACTG 381 5′- disrupted N/A N/A N/A GCGCGCATTTGTT ATGCACAAGCTAACCGATCCGCAG nifL gene/ ATGCCAATCCGG CCTATGTCGAAG GCGCGCATTTGT Prm1.2CATTCTGCCGCCA AATTAACTAAAA TATGCCAATCCG GACGGGTTTTGC AACTGCAAGATGGCATTCTGCCGC ACTTGAGACACTT CAGGCATTCGCG CAGACGGGTTTT TTGGGCGAGAACTTAAAGCCGACT GCACTTGAGACA CACCGTCTGCTGG TGAGAAATGAGA CTTTTGGGCGAG AGATAACCACCGTCTG CTGG/ TGAACATCACTG ATGCACAAGCTA CCTATGTCGAAG AATTAACTAAAAAACTGCAAGATG CAGGCATTCGCG TTAAAGCCGACT TGAGAAATGAGA AGAT-3′ CI019 ds175down 314 CGGGAACCGGTG 348 CCGTCTCTGAAG 382 5′- Prm3.1/ SEQ SEQ SEQTTATAATGCCGCG CTCTCGGTGAAC CGGGAACCGGTG disrupted ID ID ID CCCTCATATTGTGATTGTTGCGAGG TTATAATGCCGC nifL gene NO: NO: NO: GGGATTTCTTAATCAGGATGCGAGC GCCCTCATATTG 408 409 410/ GACCTATCCTGG TGGTTGTGTTTTGTGGGGATTTCTT CGCC GGCA 56- GTCCTAAAGTTGT ACATTACCGATA AATGACCTATCC CTCATAAC FAM/ AGTTGACATTAG ATGTGCCGCGTG TGGGTCCTAAAG TATT GCAC TACGGAGCACTAAC AACGGGTGCGTT TTGTAGTTGACA GTGG CCGT ACC ATG TTAGCGGAGCACGGAT TCA CGT TAAC/ C/ CCGTCTCTGAAG ZEN/ CTCTCGGTGAAC TCTG ATTGTTGCGAGGAAG CAGGATGCGAGC CTC TGGTTGTGTTTT TCG GACATTACCGAT GT/ AATGTGCCGCGT 3IABGAACGGGTGCGT kFQ/ TATG-3′ CI019 ds175 up 315 ACCGATCCGCAG 349TACAGTAGCGCC 383 5′- disrupted N/A N/A N/A GCGCGCATTTGTT TCTCAAAAATAGACCGATCCGCAG nifL gene/ ATGCCAATCCGG ATAAACGGCTCA GCGCGCATTTGT Prm3.1CATTCTGCCGCCA TGTACGTGGGCC TATGCCAATCCG GACGGGTTTTGC GTTTATTTTTTCTGCATTCTGCCGC ACTTGAGACACTT ACCCATAATCGG CAGACGGGTTTT TTGGGCGAGAACGAACCGGTGTTA GCACTTGAGACA CACCGTCTGCTGG TAATGCCGCGCC CTTTTGGGCGAG CTCAACCACCGTCTG CTGG/ TACAGTAGCGCC TCTCAAAAATAG ATAAACGGCTCA TGTACGTGGGCCGTTTATTTTTTC TACCCATAATCG GGAACCGGTGTT ATAATGCCGCGC CCTC-3′ CI006 ds20down 316 TCAACCTAAAAA 350 AACTCACTTCAC 384 5′- Prm1/ SEQ SEQ SEQAGTTTGTGTAATA ACCCCGAAGGGG TCAACCTAAAAA disrupted ID ID ID CTTGTAACGCTACGAAGTTGCCTGA AGTTTGTGTAAT nifL gene NO: NO: NO: ATGGAGATTAACCCCTACGATTCCC ACTTGTAACGCT 411 412 413 TCAATCTAGAGG GCTATTTCATTCAACATGGAGATTA TAAA CAAA /56- GTATTAATAATG CTGACCGGAGGT ACTCAATCTAGA CTGGTCGA FAM/ AATCGTACTAAA TCAAAATGACCC GGGTATTAATAA TACT AGCG AAGCTGGTACTGGGC AGCGAACCGAGT TGAATCGTACTA GGGC CCAG TTGC GC CG AACTGGTACTGGGCAA ACGG CT/ GCGC/ CT TAT ZEN/ AACTCACTTCAC GACC ACCCCGAAGGGG CTACGAAGTTGCCTGA GATT CCCTACGATTCC CCC/ CGCTATTTCATT 3IAB CACTGACCGGAG kFQ/GTTCAAAATGAC CCAGCGAACCGA GTCG-3′ CI006 ds20 up 317 GGGCGACAAACG 351CGTCCTGTAATA 385 5′- disrupted N/A N/A N/A GCCTGGTGGCAC ATAACCGGACAAGGGCGACAAACG nifL gene/ AAATTGTCAGAA TTCGGACTGATTA GCCTGGTGGCAC Prm1CTACGACACGAC AAAAAGCGCCCT AAATTGTCAGAA TAACTGACCGCA TGTGGCGCTTTTTCTACGACACGAC GGAGTGTGCGAT TTATATTCCCGCC TAACTGACCGCA GACCCTGAATATTCCATTTAAAATA GGAGTGTGCGAT GATGATGGATGC AAAAATCCAATC GACCCTGAATAT CGGCGATGATGGATGC CGGC/ CGTCCTGTAATA ATAACCGGACAA TTCGGACTGATT AAAAAAGCGCCCTTGTGGCGCTTT TTTTATATTCCC GCCTCCATTTAA AATAAAAAATCC AATC-3′ CI006 ds24up 318 GGGCGACAAACG 352 GGACATCATCGC 386 5′- disrupted SEQ SEQ SEQGCCTGGTGGCAC GACAAACAATAT GGGCGACAAACG nifL gene/ ID ID ID AAATTGTCAGAATAATACCGGCAA GCCTGGTGGCAC Prm5 NO: NO: NO: CTACGACACGAC CCACACCGGCAAAAATTGTCAGAA 414 415 416 TAACTGACCGCA TTTACGAGACTG CTACGACACGAC GGTGGCGC /56- GGAGTGTGCGAT CGCAGGCATCCT TAACTGACCGCA CACT AGTC FAM/GACCCTGAATAT TTCTCCCGTCAAT GGAGTGTGCGAT CTTT TCGT CA GATGATGGATGCTTCTGTCAAATAA GACCCTGAATAT GCAT AAAT GGA CGGC AG GATGATGGATGC GGTT TGCCGTG CGGC/ T/ GGACATCATCGC ZEN/ GACAAACAATAT GCGA TAATACCGGCAA TGACCACACCGGCAA CCC TTTACGAGACTG TGA CGCAGGCATCCT AT/ TTCTCCCGTCAA 3IABTTTCTGTCAAAT kFQ AAAG-3′ CI006 ds24 down 319 TAAGAATTATCTG 353AACTCACTTCAC 387 5′- Prm5/ N/A N/A N/A GATGAATGTGCC ACCCCGAAGGGGTAAGAATTATCT disrupted ATTAAATGCGCA GAAGTTGCCTGA GGATGAATGTGC nifL geneGCATAATGGTGC CCCTACGATTCCC CATTAAATGCGC GTTGTGCGGGAA GCTATTTCATTCAAGCATAATGGTG AACTGCTTTTTTT CTGACCGGAGGT CGTTGTGCGGGA TGAAAGGGTTGGTCAAAATGACCC AAACTGCTTTTT TCAGTAGCGGAA AGCGAACCGAGT TTTGAAAGGGTT AC CGGGTCAGTAGCGG AAAC/ AACTCACTTCAC ACCCCGAAGGGG GAAGTTGCCTGA CCCTACGATTCCCGCTATTTCATT CACTGACCGGAG GTTCAAAATGAC CCAGCGAACCGA GTCG-3′ CI006 ds30N/A 320 CGCCAGAGAGTC 354 TTTAACGATCTGA 388 5′- 5′ UTR N/A N/A N/AGAAATCGAACAT TTGGCGATGATG CGCCAGAGAGTC and ATG/ TTCCGTAATACCGAAACGGATTCGC GAAATCGAACAT truncated CGATTACCCAGG CGGAAGATGCGCTTCCGTAATACC glnE gene AGCCGTTCTGGTT TTTCTGAGAGCTG GCGATTACCCAGGCACAGCGGAAA GCGCGAATTGTG GAGCCGTTCTGG ACGTTAACGAAA GCAGGATGCGTTTTGCACAGCGGA GGATATTTCGCAT GCAGGAGGAGGA AAACGTTAACGA G TT AAGGATATTTCGCATG/ TTTAACGATCTG ATTGGCGATGAT GAAACGGATTCG CCGGAAGATGCG CTTTCTGAGAGCTGGCGCGAATTG TGGCAGGATGCG TTGCAGGAGGAG GATT-3′ CI006 ds31 N/A 321CGCCAGAGAGTC 355 GCACTGAAACAC 389 5′- 5′ UTR N/A N/A N/A GAAATCGAACATCTCATTTCCCTGT CGCCAGAGAGTC and ATG/ TTCCGTAATACCG GTGCCGCGTCGCGAAATCGAACAT truncated CGATTACCCAGG CGATGGTTGCCA TTCCGTAATACC glnE geneAGCCGTTCTGGTT GTCAGCTGGCGC GCGATTACCCAG GCACAGCGGAAA GCTACCCGATCCTGAGCCGTTCTGG ACGTTAACGAAA GCTTGATGAATT TTGCACAGCGGA GGATATTTCGCATGCTCGACCCGAA AAACGTTAACGA G TA AAGGATATTTCG CATG/ GCACTGAAACACCTCATTTCCCTG TGTGCCGCGTCG CCGATGGTTGCC AGTCAGCTGGCG CGCTACCCGATCCTGCTTGATGAA TTGCTCGACCCG AATA-3′ CI019 ds34 N/A 322 GATGATGGATGC 356GCGCTCAAACAG 390 5′- 5′ UTR N/A N/A N/A TTTCTGGTTAAAC TTAATCCGTCTGTGATGATGGATGC and ATG/ GGGCAACCTCGT GTGCCGCCTCGC TTTCTGGTTAAA truncatedTAACTGACTGACT CGATGGTCGCGA CGGGCAACCTCG glnE gene AGCCTGGGCAAACACAACTTGCAC TTAACTGACTGA CTGCCCGGGCTTT GTCATCCTTTATT CTAGCCTGGGCATTTTTGCAAGGAA GCTCGATGAACT AACTGCCCGGGC TCTGATTTCATG GCTCGACCCGCGTTTTTTTTGCAA CA GGAATCTGATTT CATG/ GCGCTCAAACAG TTAATCCGTCTGTGTGCCGCCTCG CCGATGGTCGCG ACACAACTTGCA CGTCATCCTTTA TTGCTCGATGAACTGCTCGACCCG CGCA-3′ CI019 ds70 up 323 ACCGATCCGCAG 357 AGTCTGAACTCA 3915′- disrupted N/A N/A N/A GCGCGCATTTGTT TCCTGCGGCAGT ACCGATCCGCAGnifL gene/ ATGCCAATCCGG CGGTGAGACGTA GCGCGCATTTGT Prm4 CATTCTGCCGCCATTTTTGACCAAAG TATGCCAATCCG GACGGGTTTTGC AGTGATCTACAT GCATTCTGCCGCACTTGAGACACTT CACGGAATTTTGT CAGACGGGTTTT TTGGGCGAGAAC GGTTGTTGCTGCTGCACTTGAGACA CACCGTCTGCTGG TAAAAGGGCAAA CTTTTGGGCGAG T AACCACCGTCTGCTGG/ AGTCTGAACTCA TCCTGCGGCAGT CGGTGAGACGTA TTTTTGACCAAA GAGTGATCTACATCACGGAATTTT GTGGTTGTTGCT GCTTAAAAGGGC AAAT-3′ CI019 ds70 down 324CATCGGACACCA 358 CCGTCTCTGAAG 392 5′- Prm4/ N/A N/A N/A CCAGCTTACAAACTCTCGGTGAAC CATCGGACACCA disrupted TTGCCTGATTGCG ATTGTTGCGAGGCCAGCTTACAAA nifL gene GCCCCGATGGCC CAGGATGCGAGC TTGCCTGATTGCGGTATCACTGAC TGGTTGTGTTTTG GGCCCCGATGGC CGACCATTTCGTG ACATTACCGATACGGTATCACTGA CCTTATGTCATGC ATGTGCCGCGTG CCGACCATTTCG GATGGGGGCTGGAACGGGTGCGTT TGCCTTATGTCA G ATG TGCGATGGGGGC TGGG/ CCGTCTCTGAAGCTCTCGGTGAAC ATTGTTGCGAGG CAGGATGCGAGC TGGTTGTGTTTT GACATTACCGATAATGTGCCGCGT GAACGGGTGCGT TATG-3′  137 ds799 down 325 TCTTCAACAACTG 359GCCATTGAGCTG 393 5′- PinfC/ SEQ SEQ SEQ GAGGAATAAGGT GCTTCCCGACCGTCTTCAACAACT disrupted ID ID ID ATTAAAGGCGGA CAGGGCGGCACC GGAGGAATAAGGnifL gene NO: NO: NO: AAACGAGTTCAA TGCCTGACCCTGC TATTAAAGGCGG 417 418419/ ACGGCACGTCCG GTTTCCCGCTGTT AAAACGAGTTCA CTCG AGGG 56- AATCGTATCAATTAACACCCTGAC AACGGCACGTCC GCAG TGTT FAM/ GGCGAGATTCGC CGGAGGTGAAGCGAATCGTATCAA CATG AAAC AA GCCCTGGAAGTT ATGATCCCTGAA TGGCGAGATTCG GACGAGCG CGG CGC TC CGCCCTGGAAGT TAA GGAA CAC TCGC/ A G/ GCCATTGAGCTG ZEN/GCTTCCCGACCG TCCG CAGGGCGGCACC AAT TGCCTGACCCTG CGT CGTTTCCCGCTG ATCTTTAACACCCTG AA/ ACCGGAGGTGAA 3IAB GCATGATCCCTG kFQ/ AATC-3′  137 ds799up 326 TCCGGGTTCGGCT 360 AGCGTCAGGTAC 394 5′- disrupted N/A N/A N/ATACCCCGCCGCGT CGGTCATGATTC TCCGGGTTCGGC nifL gene/ TTTGCGCACGGTGACCGTGCGATTCT TTACCCCGCCGC PinfC TCGGACAATTTGT CGGTTCCCTGGA GTTTTGCGCACGCATAACTGCGAC GCGCTTCATTGGC GTGTCGGACAAT ACAGGAGTTTGC ATCCTGACCGAATTGTCATAACTG GATGACCCTGAA GAGTTCGCTGGC CGACACAGGAGT TATGATGCTCGATTCTTCCCAACCT TTGCGATGACCC G TGAATATGATGC TCGA/ AGCGTCAGGTACCGGTCATGATTC ACCGTGCGATTC TCGGTTCCCTGG AGCGCTTCATTG GCATCCTGACCGAAGAGTTCGCTG GCTTCTTCCCAA CCTG-3′  137 ds809 N/A 327 ATCGCAGCGTCTT 361GCGCTGAAGCAC 395 5′- 5′ UTR SEQ SEQ SEQ TGAATATTTCCGT CTGATCACGCTCTATCGCAGCGTCT and ATG/ ID ID ID CGCCAGGCGCTG GCGCGGCGTCGC TTGAATATTTCCtruncated NO: NO: NO: GCTGCCGAGCCG CGATGGTCGCCA GTCGCCAGGCGC glnE gene420 421 422/ TTCTGGCTGCATA GCCAGCTGGCGC TGGCTGCCGAGC GAGC GCCG 56-GTGGAAAACGAT GCCACCCGCTGC CGTTCTGGCTGC CGTT TCGG FAM/ AATTTCAGGCCATGCTGGATGAGC ATAGTGGAAAAC CTGG CTGA TTAT GGGAGCCCTTAT TGCTGGATCCCAGATAATTTCAGG CTGC TAGA GGC G ACA CCAGGGAGCCCT ATAG GG GC/ TATG/ ZEN/GCGCTGAAGCAC TGAA CTGATCACGCTC GCA TGCGCGGCGTCG CCTG CCGATGGTCGCC ATCAGCCAGCTGGCG A/ CGCCACCCGCTG 3IAB CTGCTGGATGAG kFQ/ CTGCTGGATCCC AACA-3′ 137 ds843 up 328 TCCGGGTTCGGCT 362 GCCCGCTGACCG 396 5′- disrupted N/AN/A N/A TACCCCGCCGCGT ACCAGAACTTCC TCCGGGTTCGGC nifL gene/ TTTGCGCACGGTGACCTTGGACTCG TTACCCCGCCGC Prm1.2 TCGGACAATTTGT GCTATACCCTTGGGTTTTGCGCACG CATAACTGCGAC CGTGACGGCGCG GTGTCGGACAAT ACAGGAGTTTGCCGATAACTGGGA TTGTCATAACTG GATGACCCTGAA CTACATCCCCATT CGACACAGGAGTTATGATGCTCGA CCGGTGATCTTAC TTGCGATGACCC C TGAATATGATGC TCGA/GCCCGCTGACCG ACCAGAACTTCC ACCTTGGACTCG GCTATACCCTTG GCGTGACGGCGCGCGATAACTGGG ACTACATCCCCA TTCCGGTGATCT TACC-3′  137 ds843 down 329TCACTTTTTAGCA 363 GCCATTGAGCTG 397 5′- Prm1.2/ N/A N/A N/A AAGTTGCACTGGGCTTCCCGACCG TCACTTTTTAGC disrupted ACAAAAGGTACC CAGGGCGGCACCAAAGTTGCACTG nifL gene ACAATTGGTGTA TGCCTGACCCTGC GACAAAAGGTACCTGATACTCGAC GTTTCCCGCTGTT CACAATTGGTGT ACAGCATTAGTG TAACACCCTGACACTGATACTCGA TCGATTTTTCATA CGGAGGTGAAGC CACAGCATTAGT TAAAGGTAATTTTATGATCCCTGAA GTCGATTTTTCA G TC TATAAAGGTAAT TTTG/ GCCATTGAGCTGGCTTCCCGACCG CAGGGCGGCACC TGCCTGACCCTG CGTTTCCCGCTG TTTAACACCCTGACCGGAGGTGAA GCATGATCCCTG AATC-3′  137 ds853 up 330 TCCGGGTTCGGCT 364GCTAAAGTTCTC 398 5′- disrupted N/A N/A N/A TACCCCGCCGCGT GGCTAATCGCTGTCCGGGTTCGGC nifL gene/ TTTGCGCACGGTG ATAACATTTGAC TTACCCCGCCGC Prm6.2TCGGACAATTTGT GCAATGCGCAAT GTTTTGCGCACG CATAACTGCGAC AAAAGGGCATCAGTGTCGGACAAT ACAGGAGTTTGC TTTGATGCCCTTT TTGTCATAACTG GATGACCCTGAATTGCACGCTTTCA CGACACAGGAGT TATGATGCTCGA TACCAGAACCTG TTGCGATGACCC GCTGAATATGATGC TCGA/ GCTAAAGTTCTC GGCTAATCGCTG ATAACATTTGAC GCAATGCGCAATAAAAGGGCATCA TTTGATGCCCTT TTTGCACGCTTT CATACCAGAACC TGGC-3′  137 ds853down 331 GTTCTCCTTTGCA 365 GCCATTGAGCTG 399 5′- Prm6.2/ N/A N/A N/AATAGCAGGGAAG GCTTCCCGACCG GTTCTCCTTTGC disrupted AGGCGCCAGAACCAGGGCGGCACC AATAGCAGGGAA nifL gene CGCCAGCGTTGA TGCCTGACCCTGCGAGGCGCCAGAA AGCAGTTTGAAC GTTTCCCGCTGTT CCGCCAGCGTTG GCGTTCAGTGTATTAACACCCTGAC AAGCAGTTTGAA AATCCGAAACTT CGGAGGTGAAGC CGCGTTCAGTGTAATTTCGGTTTGG ATGATCCCTGAA ATAATCCGAAAC A TC TTAATTTCGGTT TGGA/GCCATTGAGCTG GCTTCCCGACCG CAGGGCGGCACC TGCCTGACCCTG CGTTTCCCGCTGTTTAACACCCTG ACCGGAGGTGAA GCATGATCCCTG AATC-3′  137 ds857 up 332TCCGGGTTCGGCT 366 CGCCGTCCTCGC 400 5′- disrupted N/A N/A N/ATACCCCGCCGCGT AGTACCATTGCA TCCGGGTTCGGC nifL gene/ TTTGCGCACGGTGACCGACTTTACA TTACCCCGCCGC Prm8.2 TCGGACAATTTGT GCAAGAAGTGAT GTTTTGCGCACGCATAACTGCGAC TCTGGCACGCAT GTGTCGGACAAT ACAGGAGTTTGC GGAACAAATTCTTTGTCATAACTG GATGACCCTGAA TGCCAGTCGGGC CGACACAGGAGT TATGATGCTCGATTTATCCGATGAC TTGCGATGACCC GAA TGAATATGATGC TCGA/ CGCCGTCCTCGCAGTACCATTGCA ACCGACTTTACA GCAAGAAGTGAT TCTGGCACGCAT GGAACAAATTCTTGCCAGTCGGGC TTTATCCGATGA CGAA-3′  137 ds857 down 333 GATATGCCTGAA 367GCCATTGAGCTG 401 5′- Prm8.2/ N/A N/A N/A GTATTCAATTACT GCTTCCCGACCGGATATGCCTGAA disrupted TAGGCATTTACTT CAGGGCGGCACC GTATTCAATTAC nifL geneAACGCAGGCAGG TGCCTGACCCTGC TTAGGCATTTAC CAATTTTGATGCT GTTTCCCGCTGTTTTAACGCAGGCA GCCTATGAAGCG TAACACCCTGAC GGCAATTTTGAT TTTGATTCTGTACCGGAGGTGAAGC GCTGCCTATGAA TTGAGCTTGATC ATGATCCCTGAA GCGTTTGATTCT TCGTACTTGAGCTT GATC/ GCCATTGAGCTG GCTTCCCGACCG CAGGGCGGCACC TGCCTGACCCTGCGTTTCCCGCTG TTTAACACCCTG ACCGGAGGTGAA GCATGATCCCTG AATC-3′   63 ds908down 334 TGGTATTGTCAGT 368 TCTTTAGATCTCT 402 5′- PinfC/ SEQ SEQ N/ACTGAATGAAGCT CGGTCCGCCCTG TGGTATTGTCAG disrupted ID ID CTTGAAAAAGCTATGGCGGCACCT TCTGAATGAAGC nifL gene NO: NO: GAGGAAGCGGGC TGCTGACGTTACTCTTGAAAAAGC 423 424 GTCGATTTAGTAG GCCTGCCGGTAC TGAGGAAGCGGG GGAA GGGCAAATCAGTCCGA AGCAGGTTATCA CGTCGATTTAGT AACG GGAC ATGCCGAGCCGCCCGGAGGCTTAA AGAAATCAGTCC AGTT CGAG CAGTTTGTCGAAT AATGACCCAGTTGAATGCCGAGCC CAAC AGAT C ACC GCCAGTTTGTCG CGGC CTAA AATC/ TCTTTAGATCTCTCGGTCCGCCCT GATGGCGGCACC TTGCTGACGTTA CGCCTGCCGGTA CAGCAGGTTATCACCGGAGGCTTA AAATGACCCAGT TACC-3′   63 ds908 up 335 TGCAAATTGCAC 369TGAATATCACTG 403 5′- disrupted N/A N/A N/A GGTTATTCCGGGT ACTCACAAGCTATGCAAATTGCAC nifL gene/ GAGTATATGTGT CCTATGTCGAAG GGTTATTCCGGG PinfCGATTTGGGTTCCG AATTAACTAAAA TGAGTATATGTG GCATTGCGCAAT AACTGCAAGATGTGATTTGGGTTC AAAGGGGAGAAA CAGGCATTCGCG CGGCATTGCGCA GACATGAGCATCTTAAAGCCGACT ATAAAGGGGAGA ACGGCGTTATCA TGAGAAATGAGA AAGACATGAGCA GC AGATTCACGGCGTTAT CAGC/ TGAATATCACTG ACTCACAAGCTA CCTATGTCGAAG AATTAACTAAAAAACTGCAAGATG CAGGCATTCGCG TTAAAGCCGACT TGAGAAATGAGA AGAT-3′  910 ds960up 336 TCAGGGCTGCGG 370 CTGGGGTCACTG 404 5′- disrupted N/A N/A N/AATGTCGGGCGTTT GAGCGCTTTATC TCAGGGCTGCGG nifL gene/ CACAACACAAAAGGCATCCTGACC ATGTCGGGCGTT PinfC TGTTGTAAATGCG GAAGAATTTGCC TCACAACACAAAACACAGCCGGGC GGTTTCTTCCCGA ATGTTGTAAATG CTGAAACCAGGA CCTGGCTGGCCCCGACACAGCCGG GCGTGTGATGAC CTGTTCAGGTTGT GCCTGAAACCAG CTTTAATATGATGGGTGATGAATAT GAGCGTGTGATG C CA ACCTTTAATATG ATGC/ CTGGGGTCACTGGAGCGCTTTATC GGCATCCTGACC GAAGAATTTGCC GGTTTCTTCCCG ACCTGGCTGGCCCCTGTTCAGGTT GTGGTGATGAAT ATCA-3′  910 ds960 down 337 CGGAAAACGAGT 371GCAATAGAACTA 405 5′- PinfC/ N/A N/A N/A TCAAACGGCACG ACTACCCGCCCTCGGAAAACGAGT disrupted TCCGAATCGTATC GAAGGCGGTACC TCAAACGGCACG nifL geneAATGGCGAGATT TGCCTGACCCTGC TCCGAATCGTAT CGCGCCCAGGAA GATTCCCGTTATTCAATGGCGAGAT GTTCGCTTAACTG TCATTCACTGACC TCGCGCCCAGGA GTCTGGAAGGTGGGAGGCCCACGA AGTTCGCTTAAC AGCAGCTGGGTA TGACCCAGCGAC TGGTCTGGAAGG TT CTGAGCAGCTGGG TATT/ GCAATAGAACTA ACTACCCGCCCT GAAGGCGGTACC TGCCTGACCCTGCGATTCCCGTTA TTTCATTCACTG ACCGGAGGCCCA CGATGACCCAGC GACC-3′  137 ds2551up 425 CTTCGGCGGCGT 427 GCCCGCTGACCG 429 5′- 3′ end of N/A N/A N/AGAAGAAGAGTGG ACCAGAACTTCC CTTCGGCGGCGT sad TTTTGGTCGCGAG ACCTTGGACTCGGAAGAAGAGTGG (Succinate CTGTCACATTTTG GCTATACCCTTGG TTTTGGTCGCGAsemialdehyde GTCTGCACGAGTT CGTGACGGCGCG GCTGTCACATTT dehydro-CTGTAATGCGCA CGATAACTGGGA TGGTCTGCACGA genase)/ GACCGTCTGGAACTACATCCCCATT GTTCTGTAATGC Prm1.2 AGACCGTCGCTA CCGGTGATCTTACGCAGACCGTCTG A C GAAAGACCGTCG CTAA/ GCCCGCTGACCG ACCAGAACTTCCACCTTGGACTCG GCTATACCCTTG GCGTGACGGCGC GCGATAACTGGG ACTACATCCCCATTCCGGTGATCT TACC-3′  137 ds2551 down 426 TCACTTTTTAGCA 428 ATGGCGGCGGTG430 5′- Prm1.2/ N/A N/A N/A AAGTTGCACTGG ATCAATAACGCA TCACTTTTTAGC glsA2ACAAAAGGTACC ATGCTGGAAGCC AAAGTTGCACTG ACAATTGGTGTA ATTCTGGCAGAAGACAAAAGGTAC CTGATACTCGAC ATCAGGCCGCTG CACAATTGGTGT ACAGCATTAGTGATTGGCCGCGGT ACTGATACTCGA TCGATTTTTCATA AAAGTGGCGGAT CACAGCATTAGTTAAAGGTAATTTT TACATTCCGGCG GTCGATTTTTCA G CTGG TATAAAGGTAAT TTTG/ATGGCGGCGGTG ATCAATAACGCA ATGCTGGAAGCC ATTCTGGCAGAA ATCAGGCCGCTGATTGGCCGCGGT AAAGTGGCGGAT TACATTCCGGCG CTGG-3′

TABLE 31 Remodeled Non-intergeneric Microbes Strain Name Genotype SEQ IDNO CI006 16S rDNA - contig 5 62 CI006 16S rDNA - contig 8 63 CI019 16SrDNA 64 CI006 nifH 65 CI006 nifD 66 CI006 nifK 67 CI006 nifL 68 CI006nifA 69 CI019 nifH 70 CI019 nifD 71 CI019 nifK 72 CI019 nifL 73 CI019nifA 74 CI006 Prm5 with 500bp 75 flanking regions CI006 nifLA operon -upstream 76 intergenic region plus nifL and nifA CDSs CI006 nifL (AminoAcid) 77 CI006 nifA (Amino Acid) 78 CI006 glnE 79 CI006 glnE_KO1 80CI006 glnE (Amino Acid) 81 CI006 glnE_KO1 (Amino Acid) 82 CI006 GlnEATase domain 83 (Amino Acid) CM029 Prm5 inserted into nifL 84 region

TABLE 32 Remodeled Non-intergeneric Microbes Associated Novel Strain SEQJunction If Strain ID ID NO Genotype Description Applicable CI63; 63 SEQID 16S N/A N/A CI063 NO 85 CI63; 63 SEQ ID nifH N/A N/A CI063 NO 86CI63; 63 SEQ ID nifD1 1 of 2 unique genes annotated as nifD N/A CI063 NO87 in 63 genome CI63; 63 SEQ ID nifD2 2 of 2 unique genes annotated asnifD N/A CI063 NO 88 in 63 genome CI63; 63 SEQ ID nifK1 1 of 2 uniquegenes annotated as nifK N/A CI063 NO 89 in 63 genome CI63; 63 SEQ IDnifK2 2 of 2 unique genes annotated as nifK N/A CI063 NO 90 in 63 genomeCI63; 63 SEQ ID nifL N/A N/A CI063 NO 91 CI63; 63 SEQ ID nifA N/A N/ACI063 NO 92 CI63; 63 SEQ ID glnE N/A N/A CI063 NO 93 CI63; 63 SEQ IDamtB N/A N/A CI063 NO 94 CI63; 63 SEQ ID PinfC 500bp immediatelyupstream of the N/A CI063 NO 95 ATG start codon of the infC gene CI137137 SEQ ID 16S N/A N/A NO 96 CI137 137 SEQ ID nifH1 1 of 2 unique genesannotated as nifH N/A NO 97 in 137 genome CI137 137 SEQ ID nifH2 2 of 2unique genes annotated as nifH N/A NO 98 in 137 genome CI137 137 SEQ IDnifD1 1 of 2 unique genes annotated as nifD N/A NO 99 in 137 genomeCI137 137 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A NO 100in 137 genome CI137 137 SEQ ID nifK1 1 of 2 unique genes annotated asnifK N/A NO 101 in 137 genome CI137 137 SEQ ID nifK2 2 of 2 unique genesannotated as nifK N/A NO 102 in 137 genome CI137 137 SEQ ID nifL N/A N/ANO 103 CI137 137 SEQ ID nifA N/A N/A NO 104 CI137 137 SEQ ID glnE N/AN/A NO 105 CI137 137 SEQ ID PinfC 500bp immediately upstream of the N/ANO 106 TTG start codon of infC CI137 137 SEQ ID amtB N/A N/A NO 107CI137 137 SEQ ID Prm8.2 internal promoter located in nlpI gene; N/A NO108 299bp starting at 81bp after the A of the ATG of the nlpI gene CI137137 SEQ ID Prm6.2 300bp upstream of the secE gene N/A NO 109 starting at57bp upstream of the A of the ATG of secE CI137 137 SEQ ID Prm1.2 400bpimmediately upstream of the N/A NO 110 ATG of cspE gene none 728 SEQ ID16S N/A N/A NO 111 none 728 SEQ ID nifH N/A N/A NO 112 none 728 SEQ IDnifD1 1 of 2 unique genes annotated as nifD N/A NO 113 in 728 genomenone 728 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A NO 114in 728 genome none 728 SEQ ID nifK1 1 of 2 unique genes annotated asnifK N/A NO 115 in 728 genome none 728 SEQ ID nifK2 2 of 2 unique genesannotated as nifK N/A NO 116 in 728 genome none 728 SEQ ID nifL N/A N/ANO 117 none 728 SEQ ID nifA N/A N/A NO 118 none 728 SEQ ID glnE N/A N/ANO 119 none 728 SEQ ID amtB N/A N/A NO 120 none 850 SEQ ID 16S N/A N/ANO 121 none 852 SEQ ID 16S N/A N/A NO 122 none 853 SEQ ID 16S N/A N/A NO123 none 910 SEQ ID 16S N/A N/A NO 124 none 910 SEQ ID nifH N/A N/A NO125 none 910 SEQ ID Dinitrogenase iron- N/A N/A NO 126 molybdenumcofactor CDS none 910 SEQ ID nifD1 N/A N/A NO 127 none 910 SEQ ID nifD2N/A N/A NO 128 none 910 SEQ ID nifK1 N/A N/A NO 129 none 910 SEQ IDnifK2 N/A N/A NO 130 none 910 SEQ ID nifL N/A N/A NO 131 none 910 SEQ IDnifA N/A N/A NO 132 none 910 SEQ ID glnE N/A N/A NO 133 none 910 SEQ IDamtB N/A N/A NO 134 none 910 SEQ ID PinfC 498bp immediately upstream ofthe N/A NO 135 ATG of the infC gene none 1021 SEQ ID 16S N/A N/A NO 136none 1021 SEQ ID nifH N/A N/A NO 137 none 1021 SEQ ID nifD1 1 of 2unique genes annotated as nifD N/A NO 138 in 910 genome none 1021 SEQ IDnifD2 2 of 2 unique genes annotated as nifD N/A NO 139 in 910 genomenone 1021 SEQ ID nifK1 1 of 2 unique genes annotated as nifK N/A NO 140in 910 genome none 1021 SEQ ID nifK2 2 of 2 unique genes annotated asnifK N/A NO 141 in 910 genome none 1021 SEQ ID nifL N/A N/A NO 142 none1021 SEQ ID nifA N/A N/A NO 143 none 1021 SEQ ID glnE N/A N/A NO 144none 1021 SEQ ID amtB N/A N/A NO 145 none 1021 SEQ ID PinfC 500bpimmediately upstream of the N/A NO 146 ATG start codon of the infC genenone 1021 SEQ ID Prm1 348bp includes the 319bp immediately N/A NO 147upstream of the ATG start codon of the lpp gene and the first 29bp ofthe lpp gene none 1021 SEQ ID Prm7 339bp upstream of the sspA gene, N/ANO 148 ending at 46bp upstream of the ATG of the sspA gene none 1113 SEQID 16S N/A N/A NO 149 none 1113 SEQ ID nifH N/A N/A NO 150 none 1113 SEQID nifD1 1 of 2 unique genes annotated as nifD N/A NO 151 in 1113 genomenone 1113 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A NO 152in 1113 genome none 1113 SEQ ID nifK N/A N/A NO 153 none 1113 SEQ IDnifL N/A N/A NO 154 none 1113 SEQ ID nifA partial gene due to a gap inthe sequence assembly, N/A NO 155 we can only identify a partial genefrom the 1113 genome none 1113 SEQ ID glnE N/A N/A NO 156 none 1116 SEQID 16S N/A NO 157 none 1116 SEQ ID nifH N/A NO 158 none 1116 SEQ IDnifD1 1 of 2 unique genes annotated as nifD N/A NO 159 in 1116 genomenone 1116 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A NO 160in 1116 genome none 1116 SEQ ID nifK1 1 of 2 unique genes annotated asnifK N/A NO 161 in 1116 genome none 1116 SEQ ID nifK2 2 of 2 uniquegenes annotated as nifK N/A NO 162 in 1116 genome none 1116 SEQ ID nifLN/A N/A NO 163 none 1116 SEQ ID nifA N/A N/A NO 164 none 1116 SEQ IDglnE N/A N/A NO 165 none 1116 SEQ ID amtB N/A N/A NO 166 none 1293 SEQID 16S N/A N/A NO 167 none 1293 SEQ ID nifH N/A N/A NO 168 none 1293 SEQID nifD1 1 of 2 unique genes annotated as nifD N/A NO 169 in 1293 genomenone 1293 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A NO 170in 1293 genome none 1293 SEQ ID nifK 1 of 2 unique genes annotated asnifK N/A NO 171 in 1293 genome none 1293 SEQ ID nifK1 2 of 2 uniquegenes annotated as nifK N/A NO 172 in 1293 genome none 1293 SEQ ID nifAN/A N/A NO 173 none 1293 SEQ ID glnE N/A N/A NO 174 none 1293 SEQ IDamtB1 1 of 2 unique genes annotated as amtB N/A NO 175 in 1293 genomenone 1293 SEQ ID amtB2 2 of 2 unique genes annotated as amtB N/A NO 176in 1293 genome none 1021-1612  SEQ ID ΔnifL::PinfC starting at 24bpafter the A of the ATG ds1131 NO 177 start codon, 1375bp of nifL havebeen deleted and replaced with the 1021 PinfC promoter sequence none1021-1612  SEQ ID ΔnifL::PinfC with starting at 24bp after the A of theATG ds1131 NO 178 500bp flank start codon, 1375bp of nifL have beendeleted and replaced with the 1021 PinfC promoter sequence; 500bpflanking the nifL gene upstream and downstream are included none1021-1612  SEQ ID glnEΔAR-2 glnE gene with 1673bp immediately ds1133 NO179 downstream of the ATG start codon deleted, resulting in a truncatedglnE protein lacking the adenylyl-removing (AR) domain none 1021-1612 SEQ ID glnEΔAR-2 with glnE gene with 1673bp immediately ds1133 NO 180500bp flank downstream of the ATG start codon deleted, resulting in atruncated glnE protein lacking the adenylyl-removing (AR) domain; 500bpflanking the glnE gene upstream and downstream are included none1021-1615  SEQ ID ΔnifL::Prm1 starting at 24bp after the A of the ATGds1145 NO 181 start codon, 1375bp of nifL have been deleted and replacedwith the 1021 Prm1 promoter sequence none 1021-1615  SEQ ID ΔnifL::Prm1with starting at 24bp after the A of the ATG ds1145 NO 182 500bp flankstart codon, 1375bp of nifL have been deleted and replaced with the 1021rm1 promoter sequence; 500bp flanking the nifL gene upstream anddownstream are included none 1021-1615  SEQ ID glnEΔAR-2 glnE gene with1673bp immediately ds1133 NO 183 downstream of the ATG start codondeleted, resulting in a truncated glnE protein lacking theadenylyl-removing (AR) domain none 1021-1615  SEQ ID glnEΔAR-2 with glnEgene with 1673bp immediately ds1133 NO 184 500bp flank downstream of theATG start codon deleted, resulting in a truncated glnE protein lackingthe adenylyl-removing (AR) domain; 500bp flanking the glnE gene upstreamand downstream are included none 1021-1619  SEQ ID ΔnifL::Prm1 startingat 24bp after the A of the ATG ds1145 NO 185 start codon, 1375bp of nifLhave been deleted and replaced with the 1021 Prm1 promoter sequence none1021-1619  SEQ ID ΔnifL::Prm1 with starting at 24bp after the A of theATG ds1145 NO 186 500bp flank start codon, 1375bp of nifL have beendeleted and replaced with the 1021 rm1 promoter sequence; 500bp flankingthe nifL gene upstream and downstream are included none 1021-1623  SEQID glnEΔAR-2 glnE gene with 1673bp immediately ds1133 NO 187 downstreamof the ATG start codon deleted, resulting in a truncated glnE proteinlacking the adenylyl-removing (AR) domain none 1021-1623  SEQ IDglnEΔAR-2 with glnE gene with 1673bp immediately ds1133 NO 188 500bpflank downstream of the ATG start codon deleted, resulting in atruncated glnE protein lacking the adenylyl-removing (AR) domain; 500bpflanking the glnE gene upstream and downstream are included none1021-1623  SEQ ID ΔnifL::Prm7 starting at 24bp after the A of the ATGds1148 NO 189 start codon, 1375bp of nifL have been deleted and replacedwith the 1021 Prm7 promoter sequence none 1021-1623  SEQ ID ΔnifL::Prm7with starting at 24bp after the A of the ATG ds1148 NO 190 500bp flankstart codon, 1375bp of nifL have been deleted and replaced with the 1021rm7 promoter sequence; 500bp flanking the nifL gene upstream anddownstream are included none 137-1034 SEQ ID glnEΔAR-2 glnE gene with1290bp immediately ds809 NO 191 downstream of the ATG start codondeleted, resulting in a truncated glnE protein lacking theadenylyl-removing (AR) domain none 137-1034 SEQ ID glnEΔAR-2 with glnEgene with 1290bp immediately ds809 NO 192 500bp flank downstream of theATG start codon deleted, resulting in a truncated glnE protein lackingthe adenylyl-removing (AR) domain; 500bp flanking the glnE gene upstreamand downstream are included none 137-1036 SEQ ID ΔnifL::PinfC startingat 24bp after the A of the ATG ds799 NO 193 start codon, 1372bp of nifLhave been deleted and replaced with the 137 PinfC promoter sequence none137-1036 SEQ ID ΔnifL::PinfC with starting at 24bp after the A of theATG ds799 NO 194 500bp flank start codon, 1372bp of nifL have beendeleted and replaced with the 137 PinfC promoter sequence; 500bpflanking the nifL gene upstream and downstream are included none137-1314 SEQ ID glnEΔAR-2 36bp glnE gene with 1290bp immediately none NO195 deletion downstream of the ATG start codon deleted AND 36bp deletedbeginning at 1472bp downstream of the start codon, resulting in atruncated glnE protein lacking the adenylyl-removing (AR) domain none137-1314 SEQ ID glnEΔAR-2 36bp glnE gene with 1290bp immediately none NO196 deletion downstream of the ATG start codon deleted AND 36bp deletedbeginning at 1472bp downstream of the start codon, resulting in atruncated glnE protein lacking the adenylyl-removing (AR) domain; 500bpflanking the nifL gene upstream and downstream are included none137-1314 SEQ ID ΔnifL::Prm8.2 starting at 24bp after the A of the ATGds857 NO 197 start codon, 1372bp of nifL have been deleted and replacedwith the 137 Prm8.2 promoter sequence none 137-1314 SEQ ID ΔnifL::Prm8.2with starting at 24bp after the A of the ATG ds857 NO 198 500bp flankstart codon, 1372bp of nifL have been deleted and replaced with the 137Prm8.2 promoter sequence; 500bp flanking the nifL gene upstream anddownstream are included none 137-1329 SEQ ID glnEΔAR-2 36bp glnE genewith 1290bp immediately none NO 199 deletion downstream of the ATG startcodon deleted AND 36bp deleted beginning at 1472bp downstream of thestart codon, resulting in a truncated glnE protein lacking theadenylyl-removing (AR) domain none 137-1329 SEQ ID glnEΔAR-2 36bp glnEgene with 1290bp immediately none NO 200 deletion downstream of the ATGstart codon deleted AND 36bp deleted beginning at 1472bp downstream ofthe start codon, resulting in a truncated glnE protein lacking theadenylyl-removing (AR) domain; 500bp flanking the nifL gene upstream anddownstream are included none 137-1329 SEQ ID ΔnifL::Prm6.2 starting at24bp after the A of the ATG ds853 NO 201 start codon, 1372bp of nifLhave been deleted and replaced with the 137 Prm6.2 promoter sequencenone 137-1329 SEQ ID ΔnifL::Prm6.2 with starting at 24bp after the A ofthe ATG ds853 NO 202 500bp flank start codon, 1372bp of nifL have beendeleted and replaced with the 137 Prm6.2 promoter sequence; 500bpflanking the nifL gene upstream and downstream are included none137-1382 SEQ ID ΔnifL::Prm1.2 starting at 24bp after the A of the ATGds843 NO 203 start codon, 1372bp of nifL have been deleted and replacedwith the 137 Prm1.2 promoter sequence none 137-1382 SEQ ID ΔnifL::Prm1.2with starting at 24bp after the A of the ATG ds843 NO 204 500bp flankstart codon, 1372bp of nifL have been deleted and replaced with the 137Prm1.2 promoter sequence; 500bp flanking the nifL gene upstream anddownstream are included none 137-1382 SEQ ID glnEΔAR-2 36bp glnE genewith 1290bp immediately none NO 205 deletion downstream of the ATG startcodon deleted AND 36bp deleted beginning at 1472bp downstream of thestart codon, resulting in a truncated glnE protein lacking theadenylyl-removing (AR) domain none 137-1382 SEQ ID glnEΔAR-2 36bp glnEgene with 1290bp immediately none NO 206 deletion downstream of the ATGstart codon deleted AND 36bp deleted beginning at 1472bp downstream ofthe start codon, resulting in a truncated glnE protein lacking theadenylyl-removing (AR) domain; 500bp flanking the nifL gene upstream anddownstream are included none 137-1586 SEQ ID ΔnifL::PinfC starting at24bp after the A of the ATG ds799 NO 207 start codon, 1372bp of nifLhave been deleted and replaced with the 137 PinfC promoter sequence none137-1586 SEQ ID ΔnifL::PinfC with starting at 24bp after the A of theATG ds799 NO 208 500bp flank start codon, 1372bp of nifL have beendeleted and replaced with the 137 PinfC promoter sequence; 500bpflanking the nifL gene upstream and downstream are included none137-1586 SEQ ID glnEΔAR-2 glnE gene with 1290bp immediately ds809 NO 209downstream of the ATG start codon deleted, resulting in a truncated glnEprotein lacking the adenylyl-removing (AR) domain none 137-1586 SEQ IDglnEΔAR-2 with glnE gene with 1290bp immediately ds809 NO 210 500bpflank downstream of the ATG start codon deleted, resulting in atruncated glnE protein lacking the adenylyl-removing (AR) domain; 500bpflanking the glnE gene upstream and downstream are included none 19-594SEQ ID glnEΔAR-2 glnE gene with 1650bp immediately ds34 NO 211downstream of the ATG start codon deleted, resulting in a truncated glnEprotein lacking the adenylyl-removing (AR) domain none 19-594 SEQ IDglnEΔAR-2 with glnE gene with 1650bp immediately ds34 NO 212 500bp flankdownstream of the ATG start codon deleted, resulting in a truncated glnEprotein lacking the adenylyl-removing (AR) domain; 500bp flanking theglnE gene upstream and downstream are included none 19-594 SEQ IDΔnifL::Prm6.1 starting at 221bp after the A of the ds180 NO 213 ATGstart codon, 845bp of nifL have been deleted and replaced with the CI019Prm6.1 promoter sequence none 19-594 SEQ ID ΔnifL::Prm6.1 with startingat 221bp after the A of the ds180 NO 214 500bp flank ATG start codon,845bp of nifL have been deleted and replaced with the CI019Prm6.1promoter sequence; 500bp flanking the nifL gene upstream anddownstream are included none 19-714 SEQ ID ΔnifL::Prm6.1 starting at221bp after the A of the ds180 NO 215 ATG start codon, 845bp of nifLhave been deleted and replaced with the CI019 Prm6.1 promoter sequencenone 19-714 SEQ ID ΔnifL::Prm6.1 with starting at 221bp after the A ofthe ds180 NO 216 500bp flank ATG start codon, 845bp of nifL have beendeleted and replaced with the CI019 Prm6.1promoter sequence; 500bpflanking the nifL gene upstream and downstream are included none 19-715SEQ ID ΔnifL::Prm7.1 starting at 221bp after the A of the ds181 NO 217ATG start codon, 845bp of nifL have been deleted and replaced with theCI019 Prm7.1 promoter sequence none 19-715 SEQ ID ΔnifL::Prm7.1 withstarting at 221bp after the A of the ds181 NO 218 500bp flank ATG startcodon, 845bp of nifL have been deleted and replaced with the CI019Prm76.1promoter sequence; 500bp flanking the nifL gene upstream anddownstream are included 19-713 19-750 SEQ ID ΔnifL::Prm1.2 starting at221bp after the A of the ds172 NO 219 ATG start codon, 845bp of nifLhave been deleted and replaced with the CI019 Prm1.2 promoter sequence19-713 19-750 SEQ ID ΔnifL::Prm1.2 with starting at 221bp after the A ofthe ds172 NO 220 500bp flank ATG start codon, 845bp of nifL have beendeleted and replaced with the CI019 Prm1.2 promoter sequence; 500bpflanking the nifL gene upstream and downstream are included 17-72419-804 SEQ ID ΔnifL::Prm1.2 starting at 221bp after the A of the ds172NO 221 ATG start codon, 845bp of nifL have been deleted and replacedwith the CI019 Prm1.2 promoter sequence 17-724 19-804 SEQ IDΔnifL::Prm1.2 with starting at 221bp after the A of the ds172 NO 222500bp flank ATG start codon, 845bp of nifL have been deleted andreplaced with the CI019 Prm1.2 promoter sequence; 500bp flanking thenifL gene upstream and downstream are included 17-724 19-804 SEQ IDglnEΔAR-2 glnE gene with 1650bp immediately ds34 NO 223 downstream ofthe ATG start codon deleted, resulting in a truncated glnE proteinlacking the adenylyl-removing (AR) domain 17-724 19-804 SEQ ID glnEΔAR-2with glnE gene with 1650bp immediately ds34 NO 224 500bp flankdownstream of the ATG start codon deleted, resulting in a truncated glnEprotein lacking the adenylyl-removing (AR) domain; 500bp flanking theglnE gene upstream and downstream are included 19-590 19-806 SEQ IDΔnifL::Prm3.1 starting at 221bp after the A of the ds175 NO 225 ATGstart codon, 845bp of nifL have been deleted and replaced with the CI019Prm3.1 promoter sequence 19-590 19-806 SEQ ID ΔnifL::Prm3.1 withstarting at 221bp after the A of the ds175 NO 226 500bp flank ATG startcodon, 845bp of nifL have been deleted and replaced with the CI019Prm3.1 promoter sequence; 500bp flanking the nifL gene upstream anddownstream are included 19-590 19-806 SEQ ID glnEΔAR-2 glnE gene with1650bp immediately ds34 NO 227 downstream of the ATG start codondeleted, resulting in a truncated glnE protein lacking theadenylyl-removing (AR) domain 19-590 19-806 SEQ ID glnEΔAR-2 with glnEgene with 1650bp immediately ds34 NO 228 500bp flank downstream of theATG start codon deleted, resulting in a truncated glnE protein lackingthe adenylyl-removing (AR) domain; 500bp flanking the glnE gene upstreamand downstream are included none  63-1146 SEQ ID ΔnifL::PinfC startingat 24bp after the A of the ATG ds908 NO 229 start codon, 1375bp of nifLhave been deleted and replaced with the 63 PinfC promoter sequence none 63-1146 SEQ ID ΔnifL::PinfC with starting at 24bp after the A of theATG ds908 NO 230 500bp flank start codon, 1375bp of nifL have beendeleted and replaced with the 63 PinfC promoter sequence; 500bp flankingthe nifL gene upstream and downstream are included CM015;  6-397 SEQ IDΔnifL::Prm5 starting at 31bp after the A of the ATG ds24 PBC6.15 NO 231start codon, 1375bp of nifL have been deleted and replaced with theCI006 Prm5 promoter sequence CM015;  6-397 SEQ ID ΔnifL::Prm5 withstarting at 31bp after the A of the ATG ds24 PBC6.15 NO 232 500bp flankstart codon, 1375bp of nifL have been deleted and replaced with theCI006 Prm5 promoter sequence; 500bp flanking the nifL gene upstream anddownstream are included CM014  6-400 SEQ ID ΔnifL::Prm1 starting at 31bpafter the A of the ATG ds20 NO 233 start codon, 1375bp of nifL have beendeleted and replaced with the CI006 Prm1 promoter sequence CM014  6-400SEQ ID ΔnifL::Prm1 with starting at 31bp after the A of the ATG ds20 NO234 500bp flank start codon, 1375bp of nifL have been deleted andreplaced with the CI006 Prm1 promoter sequence; 500bp flanking the nifLgene upstream and downstream are included CM037;  6-403 SEQ IDΔnifL::Prm1 starting at 31bp after the A of the ATG ds20 PBC6.37 NO 235start codon, 1375bp of nifL have been deleted and replaced with theCI006 Prm1 promoter sequence CM037;  6-403 SEQ ID ΔnifL::Prm1 withstarting at 31bp after the A of the ATG ds20 PBC6.38 NO 236 500bp flankstart codon, 1375bp of nifL have been deleted and replaced with theCI006 Prm1 promoter sequence; 500bp flanking the nifL gene upstream anddownstream are included CM037;  6-403 SEQ ID glnEΔAR-2 glnE gene with1644bp immediately ds31 PBC6.39 NO 237 downstream of the ATG start codondeleted, resulting in a truncated glnE protein lacking theadenylyl-removing (AR) domain CM037;  6-403 SEQ ID glnEΔAR-2 with glnEgene with 1644bp immediately ds31 PBC6.40 NO 238 500bp flank downstreamof the ATG start codon deleted, resulting in a truncated glnE proteinlacking the adenylyl-removing (AR) domain; 500bp flanking the glnE geneupstream and downstream are included CM038;  6-404 SEQ ID glnEΔAR-1 glnEgene with 1287bp immediately ds30 PBC6.38 NO 239 downstream of the ATGstart codon deleted, resulting in a truncated glnE protein lacking theadenylyl-removing (AR) domain CM038;  6-404 SEQ ID ΔnifL::Prm1 startingat 31bp after the A of the ATG ds20 PBC6.38 NO 240 start codon, 1375bpof nifL have been deleted and replaced with the CI006 Prm1 promotersequence CM038;  6-404 SEQ ID ΔnifL::Prm1 with starting at 31bp afterthe A of the ATG ds20 PBC6.38 NO 241 500bp flank start codon, 1375bp ofnifL have been deleted and replaced with the CI006 Prm1 promotersequence; 500bp flanking the nifL gene upstream and downstream areincluded CM038;  6-404 SEQ ID glnEΔAR-1 with glnE gene with 1287bpimmediately ds30 PBC6.38 NO 242 500bp flank downstream of the ATG startcodon deleted, resulting in a truncated glnE protein lacking theadenylyl-removing (AR) domain; 500bp flanking the glnE gene upstream anddownstream are included CM029;  6-412 SEQ ID glnEΔAR-1 glnE gene with1287bp immediately ds30 PBC6.29 NO 243 downstream of the ATG start codondeleted, resulting in a truncated glnE protein lacking theadenylyl-removing (AR) domain CM029;  6-412 SEQ ID glnEΔAR-1 with glnEgene with 1287bp immediately ds30 PBC6.29 NO 244 500bp flank downstreamof the ATG start codon deleted, resulting in a truncated glnE proteinlacking the adenylyl-removing (AR) domain; 500bp flanking the glnE geneupstream and downstream are included CM029;  6-412 SEQ ID ΔnifL::Prm5starting at 31bp after the A of the ATG ds24 PBC6.29 NO 245 start codon,1375bp of nifL have been deleted and replaced with the CI006 Prm5promoter sequence CM029;  6-412 SEQ ID ΔnifL::Prm5 with starting at 31bpafter the A of the ATG ds24 PBC6.29 NO 246 500bp flank start codon,1375bp of nifL have been deleted and replaced with the CI006 Prm5promoter sequence; 500bp flanking the nifL gene upstream and downstreamare included CM093;  6-848 SEQ ID ΔnifL::Prm1 starting at 31bp after theA of the ATG ds20 PBC6.93 NO 247 start codon, 1375bp of nifL have beendeleted and replaced with the CI006 Prm1 promoter sequence CM093;  6-848SEQ ID ΔnifL::Prm1 with starting at 31bp after the A of the ATG ds20PBC6.93 NO 248 500bp flank start codon, 1375bp of nifL have been deletedand replaced with the CI006 Prm1 promoter sequence; 500bp flanking thenifL gene upstream and downstream are included CM093;  6-848 SEQ IDglnEΔAR-2 glnE gene with 1644bp immediately ds31 PBC6.93 NO 249downstream of the ATG start codon deleted, resulting in a truncated glnEprotein lacking the adenylyl-removing (AR) domain CM093;  6-848 SEQ IDglnEΔAR-2 with glnE gene with 1644bp immediately ds31 PBC6.93 NO 250500bp flank downstream of the ATG start codon deleted, resulting in atruncated glnE protein lacking the adenylyl-removing (AR) domain; 500bpflanking the glnE gene upstream and downstream are included CM093; 6-848 SEQ ID ΔamtB First 1088bp of amtB gene and 4bp ds126 PBC6.93 NO251 upstream of start codon deleted; 199bp of gene remaining lacks astart codon; no amtB protein is translated CM093;  6-848 SEQ ID ΔamtBwith 500bp First 1088bp of amtB gene and 4bp ds126 PBC6.93 NO 252 flankupstream of start codon deleted; 199bp of gene remaining lacks a startcodon; no amtB protein is translated CM094:  6-881 SEQ ID glnEΔAR-1 glnEgene with 1287bp immediately ds30 PBC6.94 NO 253 downstream of the ATGstart codon deleted, resulting in a truncated glnE protein lacking theadenylyl-removing (AR) domain CM094;  6-881 SEQ ID glnEΔAR-1 with glnEgene with 1287bp immediately ds30 PBC6.94 NO 254 500bp flank downstreamof the ATG start codon deleted, resulting in a truncated glnE proteinlacking the adenylyl-removing (AR) domain; 500bp flanking the glnE geneupstream and downstream are included CM094;  6-881 SEQ ID ΔnifL::Prm1starting at 31bp after the A of the ATG ds20 PBC6.94 NO 255 start codon,1375bp of nifL have been deleted and replaced with the CI006 Prm1promoter sequence CM094;  6-881 SEQ ID ΔnifL::Prm1 with starting at 31bpafter the A of the ATG ds20 PBC6.94 NO 256 500bp flank start codon,1375bp of nifL have been deleted and replaced with the CI006 Prm1promoter sequence; 500bp flanking the nifL gene upstream and downstreamare included CM094;  6-881 SEQ ID ΔamtB First 1088bp of amtB gene and4bp ds126 PBC6.94 NO 257 upstream of start codon deleted; 199bp of generemaining lacks a start codon; no amtB protein is translated CM094; 6-881 SEQ ID ΔamtB with 500bp First 1088bp of amtB gene and 4bp ds126PBC6.94 NO 258 flank upstream of start codon deleted; 199bp of generemaining lacks a start codon; no amtB protein is translated none910-1246 SEQ ID ΔnifL::PinfC starting at 20bp after the A of the ATGds960 NO 259 start codon, 1379bp of nifL have been deleted and replacedwith the 910 PinfC promoter sequence none 910-1246 SEQ ID ΔnifL::PinfCwith starting at 20bp after the A of the ATG ds960 NO 260 500bp flankstart codon, 1379bp of nifL have been deleted and replaced with the 910PinfC promoter sequence; 500bp flanking the nifL gene upstream anddownstream are included PBC6.1, CI006 SEQ ID 16S-1 1 of 3 unique 16SrDNA genes in the N/A 6, CI6 NO 261 CI006 genome PBC6.1, CI006 SEQ ID16S-2 2 of 3 unique 16S rDNA genes in the N/A 6. CI6 NO 262 CI006 genomePBC6.1. CI006 SEQ ID nifH N/A N/A 6, CI6 NO 263 PBC6.1, CI006 SEQ IDnifD2 2 of 2 unique genes annotated as nifD N/A 6, CI6 NO 264 in CI006genome PBC6.1, CI006 SEQ ID nifK2 2 of 2 unique genes annotated as nifKN/A 6. CI6 NO 265 in CI006 genome PBC6.1, CI006 SEQ ID nifL N/A N/A 6,06NO 266 PBC6.1. CI006 SEQ ID nifA N/A N/A 6, CI6 NO 267 PBC6.1, CI006 SEQID glnE N/A N/A 6, CI6 NO 268 PBC6.1, CI006 SEQ ID 16S-3 3 of 3 unique16S rDNA genes in the N/A 6,06 NO 269 CI006 genome PBC6.1, CI006 SEQ IDnifD1 1 of 2 unique genes annotated as nifD N/A 6, CI6 NO 270 in CI006genome PBC6.1. CI006 SEQ ID nifK1 1 of 2 unique genes annotated as nifKN/A 6, CI6 NO 271 in CI006 genome PBC6.1, CI006 SEQ ID amtB N/A N/A 6,CI6 NO 272 PBC6.1, CI006 SEQ ID Prm1 348bp includes the 319bpimmediately N/A 6, CI6 NO 273 upstream of the ATG start codon of the lppgene and the first 29bp of the lpp gene PBC6.1, CI006 SEQ ID Prm5 313bpstarting at 432bp upstream of the N/A 6, CI6 NO 274 ATG start codon ofthe ompX gene and ending 119bp upstream of the ATG start codon of theompX gene 19, CI19 CI019 SEQ ID nifL N/A N/A NO 275 19, CI19 CI019 SEQID nifA N/A N/A NO 276 19, CI19 CI019 SEQ ID 16S-1 1 of 7 unique 16SrDNA genes in the N/A NO 277 CI019 genome 19, CI19 CI019 SEQ ID 16S-2 2of 7 unique 16S rDNA genes in the N/A NO 278 CI019 genome 19, CI19 CI019SEQ ID 16S-3 3 of 7 unique 16S rDNA genes in the N/A NO 279 CI019 genome19, CI19 CI019 SEQ ID 16S-4 4 of 7 unique 16S rDNA genes in the N/A NO280 CI019 genome 19, CI19 CI019 SEQ ID 16S-5 5 of 7 unique 16S rDNAgenes in the N/A NO 281 CI019 genome 19, CI19 CI019 SEQ ID 16S-6 6 of 7unique 16S rDNA genes in the N/A NO 282 CI019 genome 19, CI19 CI019 SEQID 16S-7 7 of 7 unique 16S rDNA genes in the N/A NO 283 CI019 genome 19,CI19 CI019 SEQ ID nifH1 1 of 2 unique genes annotated as nifH N/A NO 284in CI019 genome 19, CI19 CI019 SEQ ID nifH2 2 of 2 unique genesannotated as nifH N/A NO 285 in CI019 genome 19, CI19 CI019 SEQ ID nifD11 of 2 unique genes annotated as nifD N/A NO 286 in CI019 genome 19,CI19 CI019 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A NO 287in CI019 genome 19, CI19 CI019 SEQ ID nifK1 1 of 2 unique genesannotated as nifK N/A NO 288 in CI019 genome 19, CI19 CI019 SEQ ID nifK22 of 2 unique genes annotated as nifK N/A NO 289 in CI019 genome 19,CI19 CI019 SEQ ID glnE N/A N/A NO 290 19, CI19 CI019 SEQ ID Prm4 449bpimmediately upstream of the N/A NO 291 ATG of the dscC 2 gene 19, CI19CI019 SEQ ID Prm1.2 500bp immediately upstream of the N/A NO 292 TTGstart codon of the infC gene 19, CI19 CI019 SEQ ID Prm3.1 170 bpimmediately upstream of the N/A NO 293 ATG start codon of the rplN gene19, CI20 CI020 SEQ ID Prm6.1 142bp immediately upstream of the N/A NO294 ATG of a highly-expressed hypothetical protein (annotated asPROKKA_00662 in CI019 assembly 82) 19, CI21 CI021 SEQ ID Prm7.1 293bpimmediately upstream of the N/A NO 295 ATG of the lpp gene 19-375, CM67SEQ ID glnEΔAR-2 glnE gene with 1650bp immediately ds34 19-417, NO 296downstream of the ATG start codon CM067 deleted, resulting in atruncated glnE protein lacking the adenylyl-removing (AR) domain 19-375,CM67 SEQ ID glnEΔAR-2 with glnE gene with 1650bp immediately ds3419-417, NO 297 500bp flank downstream of the ATG start codon CM067deleted, resulting in a truncated glnE protein lacking theadenylyl-removing (AR) domain; 500bp flanking the glnE gene upstream anddownstream are included 19-375, CM67 SEQ ID ΔnifL::null-v1 starting at221bp after the A of the none 19-417, NO 298 ATG start codon, 845bp ofnifL have CM067 been deleted and replaced with the 31bp sequence“GGAGTCTGAACTCATCCTGCGATGGGGGCTG” 19-375, CM67 SEQ ID ΔnifL::null-v1with starting at 221bp after the A of the none 19-417, NO 299 500bpflank ATG start codon, 845bp of nifL have CM067 been deleted andreplaced with the 31bp sequence “GGAGTCTGAACTCATCCTGCGATGGGGGCTG”; 500bpflanking the nifL gene upstream and downstream are included 19-377, CM69SEQ ID ΔnifL::null-v2 starting at 221bp after the A of the none CM069 NO300 ATG start codon, 845bp of nifL have been deleted and replaced withthe 5bp sequence “TTAAA” 19-377, CM69 SEQ ID ΔnifL::null-v2 withstarting at 221bp after the A of the none CM069 NO 301 500bp flank ATGstart codon, 845bp of nifL have been deleted and replaced with the 5bpsequence “TTAAA”; 500bp flanking the nifL gene upstream and downstreamare included 19-389, CM81 SEQ ID ΔnifL::Prm4 starting at 221bp after theA of the ds70 19-418, NO 302 ATG start codon, 845bp of nifL have CM081been deleted and replaced with the CI19 Prm4 sequence 19-389, CM81 SEQID ΔnifL::Prm4 with starting at 221bp after the A of the ds70 19-418, NO303 500bp flank ATG start codon, 845bp of nifL have CM081 been deletedand replaced with the CI19 Prm4 sequence; 500bp flanking the nifL geneupstream and downstream are included 137-2084 SEQ ID glnE_KO2 glnE genewith 1290bp immediately ds809 NO 191 downstream of the ATG start codondeleted, resulting in a truncated glnE protein lacking theadenylyl-removing (AR) domain 137-2084 SEQ ID glnE_KO2 with glnE genewith 1290bp immediately ds809 NO 192 500bp flank downstream of the ATGstart codon deleted, resulting in a truncated glnE protein lacking theadenylyl-removing (AR) domain; 500bp flanking the glnE gene upstream anddownstream are included 137-2084 SEQ ID ΔnifL-Prm1.2 starting at 24bpafter the A of the ATG ds843 NO 203 start codon, 1372bp of nifL havebeen deleted and replaced with the 137 Prm1.2 promoter sequence 137-2084SEQ ID ΔnifL-Prm1.2 with starting at 24bp after the A of the ATG ds843NO 204 500bp flank start codon, 1372bp of nifL have been deleted andreplaced with the 137 Prm1.2 promoter sequence; 500bp flanking the nifLgene upstream and downstream are included 137-2237 SEQ ID glnE_KO2 glnEgene with 1290bp immediately ds809 NO 191 downstream of the ATG startcodon deleted, resulting in a truncated glnE protein lacking theadenylyl-removing (AR) domain 137-2237 SEQ ID glnE_KO2 with glnE genewith 1290bp immediately ds809 NO 192 500bp flank downstream of the ATGstart codon deleted, resulting in a truncated glnE protein lacking theadenylyl-removing (AR) domain; 500bp flanking the glnE gene upstream anddownstream are included 137-2237 SEQ ID ΔnifL-Prm1.2 starting at 24bpafter the A of the ATG ds843 NO 203 start codon, 1372bp of nifL havebeen deleted and replaced with the 137 Prm1.2 promoter sequence 137-2237SEQ ID ΔnifL-Prm1.2 with starting at 24bp after the A of the ATG ds843NO 204 500bp flank start codon, 1372bp of nifL have been deleted andreplaced with the 137 Prm1.2 promoter sequence; 500bp flanking the nifLgene upstream and downstream are included 137-2237 SEQ IDglsA2::Prm1.2_AT 81bp immediately upstream of the ds2551 NO 431 G-startATG start codon were deleted and replaced with the 137 Prm1.2 promotersequence 137-2237 SEQ ID glsA2::Prm1.2_AT 81bp immediately upstream ofthe ds2551 NO 432 G-start with 500bp ATG start codon were deleted andflank replaced with the 137 Prm1.2 promoter sequence; 500bp flanking thePrm1.2 promoter and glsA2 gene upstream and downstream are included

Example 7: Guided Microbial Remodeling Campaign for the RationalImprovement of Multiple Phenotypic Traits

In this example, Steps A-F described in Example 1 were used to generateseveral non-transgenic derivative strains of Klebsiella variicola Wildtype (WT) strain, CI137. First, the WT strain, CI137, was isolated froma rhizosphere, characterized, and domesticated using the approachesdescribed in steps A-C of Example 1.

Then using the approaches described in steps D-F of Example 1, multiplephenotypic traits of CI137 were rationally improved without the use oftransgenes. For example, to test whether the nitrogen fixation trait ofthe WT strain can be improved, various genes involved in nitrogenfixation as described throughout this application were targeted toengineer non-intergeneric mutations, the engineered/remodeled microbeswere analyzed for nitrogen fixation, and the engineering and theanalytics steps were iterated to test whether further improvements canbe made in the nitrogen fixation ability. Once substantial improvementin the nitrogen fixation ability was achieved for a remodeled strain ofCI137, the microbes were assessed for the colonization trait. As theremodeled strains with improved nitrogen fixation showed lowercolonization than the WT strain, genes involved in colonization asdescribed in this application were targeted to engineer non-intergenericmutations to improve colonization, the engineered/remodeled microbeswere analyzed for colonization, and the engineering and the analyticssteps were iterated.

To generate non-intergeneric mutations through the iterative remodelingprocess, two approaches were employed to remodel the underlying geneticarchitecture of the microbe: (1) creating markerless deletions ofgenomic sequences encoding protein domains or whole genes, and (2)rewiring regulatory networks by intragenomic promoter rearrangement.

The remodeled strains of CI137 obtained through this iterativeremodeling process that showed improvement in nitrogen fixation and/orcolonization are described below.

To test whether the nitrogen fixation trait of the WT strain, CI137, canbe improved, a 1647 basepair region after the start codon of the glnEgene was deleted to produce a truncated GlnE protein that does notcomprise the adenylyl-removing (AR) domain resulting in a constitutivelyadenylylated glutamine synthetase. This remodeled CI137 is referred toherein as 137-1034. 137-1034 could excrete ammonia whereas the WT straindid not excrete ammonia (FIG. 26). However, 137-1034 showed reducedcolonization compared to the WT strain (FIGS. 27-28). The step ofgenerating non-intergeneric mutations was iterated to further remodel137-1034 and to test if any improvement in ammonia excretion can beobtained. For this, the nifL gene was deleted from 20 bp after the ATGstart codon to 87 bp before the TGA stop codon and a 400 bp fragmentfrom the region upstream of the cspE gene containing a promoter of thecspE gene was inserted (Prm1.2) upstream of nifA replacing the deletedportion. This remodeled microbe is referred to herein as 137-2084. Thisstrain showed improved ammonia excretion over 137-1034 (FIG. 26). Whentested for colonization potential, 137-2084 showed slightly improvedcolonization compared to 137-1034; however, its colonization potentialwas still lower by ˜10-fold compared to the WT strain (FIGS. 27-28). Thestep of generating non-intergeneric mutations was iterated/repeated totest whether both the colonization potential and ammonia excretion couldbe improved. Specifically, the nifL gene was deleted from 20 bp afterthe ATG start codon to 87 bp before the TGA stop codon; a 400 bpfragment from the region upstream of the cspE gene containing a promoterof the cspE gene was inserted (Prm1.2) upstream of nifA replacing thedeleted portion; and a 400 bp fragment from the region upstream of thecspE gene containing a promoter of the cspE gene was inserted (Prm1.2)upstream of the glsA2 gene replacing the native promoter of the gene.This strain is referred to herein as 137-2237. 137-2237 showed similarlevels of improved ammonia excretion as 137-2084 (FIG. 26) and at thesame time, the colonization potential of 137-2237 was similar orslightly improved compared to the WT strain (FIGS. 27-28).

The above description illustrates how steps D-F described in Example 1can be used to iterate non-intergeneric mutations to stack improvementsin multiple traits in remodeled microbes.

The ammonia excretion assay used here is as described in Example 2.

Colonization Assay

Briefly, strains were grown overnight in rich media (SOB). Opticaldensities of culture media containing respective strains were measured,normalized to 1 and and 1 mL of the culture medium was used to inoculatea corn seed during planting in a grow-pot. Plants were grown for 3weeks, then de-potted, and roots were washed to remove loose sand anddebris. Samples of root tissue were taken from the seminal root andfirst node (found 1-2 inches below the crown), washed in PBS, and a gDNAprep was done on the solution.

Cells per gram/fresh weight was determined by running the gDNA prepsthrough qPCR using strain specific primers. A more detailed descriptionof the assay can be found in a PCT publication, WO/2019/032926,incorporated by reference herein in its entirety.

The above-described mutations made to the C1137 WT strain are summarizedin Table 33 below.

TABLE 33 List of isolated and derivative K. variicola strains Strain IDGenotype Mutation Mutation Description 137 WT WT Wild type Klebsiellavariicola strain. 137-1034 ΔglnE_(AR)-KO2 ΔglnE_(AR)-KO2 Deletion of1647bp after the start codon of the glnE gene. 137-2084 ΔnifL::Prm1.2,ΔnifL::Prm1.2 Deletion of the nifL gene from 20bp after the ATGΔglnE_(AR)-KO2, (start) to 87bp before the TGA (stop) of the gene. A400bp fragment from the region upstream of the cspE gene containing apromoter of the cspE gene was inserted (Prm1.2) upstream of nifAreplacing the deleted portion. ΔglnE_(AR)-KO2 Deletion of 1647bp afterthe start codon of the glnE gene. 137-2337 ΔnifL::Prm1.2, ΔnifL::Prm1.2Deletion of the nifL gene from 20bp after the ATG ΔglnE_(AR)-KO2,(start) to 87bp before the TGA (stop) of the gene. glsA2::Prm1.2 A 400bpfragment from the region upstream of the cspE gene containing a promoterof the cspE gene was inserted (Prm1.2) upstream of nifA replacing thedeleted portion. ΔglnE_(AR)-KO2 Deletion of 1647bp after the start codonof the glnE gene. glsA2::Prm1.2 A 400bp fragment from the regionupstream of the cspE gene containing a promoter of the cspE gene wasinserted (Prm1.2) upstream of the glsA2 gene replacing the nativepromoter of the gene.

Numbered Embodiments of the Disclosure

Notwithstanding the appended claims, the disclosure sets forth thefollowing numbered embodiments:

-   1. A guided microbial remodeling method for the rational improvement    of plant-associated microbes to perform plant-beneficial functions,    comprising:    -   a. providing a plurality of microbial species that are        associated with a target plant of interest;    -   b. sequencing the genomes of the plurality of microbial species        and characterizing one or more genomic pathways, or sets of        genes, that are associated with a plant-beneficial function of        interest;    -   c. assaying the plurality of microbial species for: colonization        metrics, transcriptionally active genes under metabolically        relevant environmental conditions, and ability to perform a        plant-beneficial function of interest;    -   d. selecting a candidate microbial species from the plurality of        assayed microbial species;    -   e. transforming the candidate microbial species with a        transformation plasmid comprising:        -   i. a selection marker,        -   ii. a counterselection marker,        -   iii. a DNA fragment comprising: a non-intergeneric genetic            variation to be introduced into the candidate microbial            species at a target genomic locus in one or more genomic            pathways, or sets of genes, that are associated with a            plant-beneficial function of interest, and homology arms to            the target genomic locus flanking the non-intergeneric            genetic variation; and        -   iv. plasmid backbone,    -   f. selecting for a candidate microbial species that has        undergone an initial homologous recombination and has the        non-intergeneric genetic variation integrated into the target        genomic locus based on the presence of the selection marker in        the genome;    -   g. selecting for a candidate microbial species that has the        non-intergeneric genetic variation integrated into the target        genomic locus, but has undergone an additional homologous        recombination that loops-out the plasmid backbone, based on the        absence of the counterselection marker;    -   h. sequencing the genome of the transformed candidate microbial        species and confirming integration of the non-intergeneric        genetic variation at the target genomic locus and absence of any        genetic sequence from the transformation plasmid; and    -   i. repeating steps e)-h) one or more times, until a candidate        microbial species has acquired an improved ability to perform        the plant-beneficial function of interest.-   2. The guided microbial remodeling method according to embodiment 1,    wherein the plant-beneficial function of interest is nitrogen    fixation.-   3. The guided microbial remodeling method according to embodiment 1,    wherein the plant-beneficial function of interest is phosphate    solubilization.-   4. The guided microbial remodeling method according to embodiment 1,    wherein the plant-beneficial function of interest is microbial    colonization.-   5. The guided microbial remodeling method according to any one of    the embodiments 1-4, wherein step b) comprises whole genome    sequencing.-   6. The guided microbial remodeling method according to any one of    the embodiments 1-5, wherein step b) comprises characterizing the    nitrogen fixation pathway.-   7. The guided microbial remodeling method according to any one of    the embodiments 1-6, wherein step b) comprises characterizing a set    of genes selected from the group consisting of: nifA, nifL, ntrB,    ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB,    glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE,    nifJ nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW,    nifZ, nifM, nifF, nifB, nifQ, a gene associated with biosynthesis of    a nitrogenase enzyme, and combinations thereof.-   8. The guided microbial remodeling method according to any one of    the embodiments 1-7, wherein step c) comprises assaying the    plurality of microbial species for colonization metrics under    greenhouse or lab based conditions.-   9. The guided microbial remodeling method according to any one of    the embodiments 1-7, wherein step c) comprises assaying the    plurality of microbial species for colonization metrics under field    conditions.-   10. The guided microbial remodeling method according to any one of    the embodiments 1-7, wherein step c) comprises assaying the    plurality of microbial species for colonization metrics under    greenhouse or lab based conditions, and also under field conditions.-   11. The guided microbial remodeling method according to any one of    the embodiments 1-10, wherein a colonization metric assayed in    step c) comprises at least one of the following: spatial    colonization patterns, temporal colonization dynamics, density of    colonization, or combinations thereof.-   12. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs under greenhouse    or lab based conditions.-   13. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs under greenhouse    or lab based conditions and comprises measuring the transcriptomic    profile of the microbial species.-   14. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs under greenhouse    or lab based conditions and comprises measuring the transcriptomic    activity of genes associated with the microbial species ability to    perform a plant-beneficial function of interest.-   15. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs under greenhouse    or lab based conditions and comprises measuring the transcriptomic    activity of regulatory gene sequences.-   16. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs under greenhouse    or lab based conditions and comprises measuring the transcriptomic    activity of promoter sequences.-   17. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs under greenhouse    or lab based conditions and comprises measuring the transcriptomic    activity of promoter sequences in the presence of exogenous    nitrogen.-   18. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs under greenhouse    or lab based conditions and comprises measuring the transcriptomic    activity of promoter sequences in the presence of exogenous    nitrogen, wherein said transcriptomic activity of the promoter    sequences is measured by quantifying the expression of a regulated    gene.-   19. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs under field    conditions.-   20. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs under field    conditions and comprises measuring the transcriptomic profile of the    microbial species.-   21. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs under field    conditions and comprises measuring the transcriptomic activity of    genes associated with the microbial species ability to perform a    plant-beneficial function of interest.-   22. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs under field    conditions and comprises measuring the transcriptomic activity of    regulatory gene sequences.-   23. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs under field    conditions and comprises measuring the transcriptomic activity of    promoter sequences.-   24. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs under field    conditions and comprises measuring the transcriptomic activity of    promoter sequences in the presence of exogenous nitrogen.-   25. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs under field    conditions and comprises measuring the transcriptomic activity of    promoter sequences in the presence of exogenous nitrogen, wherein    said transcriptomic activity of the promoter sequences is measured    by quantifying the expression of a regulated gene.-   26. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs in vitro.-   27. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs in vitro and    comprises measuring the transcriptomic profile of the microbial    species.-   28. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs in vitro and    comprises measuring the transcriptomic activity of genes associated    with the microbial species ability to perform a plant-beneficial    function of interest.-   29. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs in vitro and    comprises measuring the transcriptomic activity of regulatory gene    sequences.-   30. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs in vitro and    comprises measuring the transcriptomic activity of promoter    sequences.-   31. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs in vitro and    comprises measuring the transcriptomic activity of promoter    sequences in N-depleted and N-replete conditions.-   32. The guided microbial remodeling method according to any one of    the embodiments 1-11, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions in step c) occurs in vitro and    comprises measuring the transcriptomic activity of promoter    sequences in N-depleted and N-replete conditions, wherein said    transcriptomic activity of the promoter sequences is measured by    quantifying the expression of a regulated gene.-   33. The guided microbial remodeling method according to any one of    the embodiments 1-7, wherein assaying the plurality of microbial    species for colonization metrics, comprises: growing said plurality    of microbial species in intimate association with a target plant.-   34. The guided microbial remodeling method according to any one of    the embodiments 1-7, wherein assaying the plurality of microbial    species for colonization metrics, comprises: growing said plurality    of microbial species in intimate association with a target plant    under greenhouse or lab based conditions.-   35. The guided microbial remodeling method according to any one of    the embodiments 1-7, wherein assaying the plurality of microbial    species for colonization metrics, comprises: growing said plurality    of microbial species in intimate association with a target plant    under field conditions.-   36. The guided microbial remodeling method according to any one of    the embodiments 1-7, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions, comprises: growing said plurality    of microbial species in intimate association with a target plant.-   37. The guided microbial remodeling method according to any one of    the embodiments 1-7, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions, comprises: growing said plurality    of microbial species in intimate association with a target plant    under greenhouse or lab based conditions.-   38. The guided microbial remodeling method according to any one of    the embodiments 1-7, wherein assaying the plurality of microbial    species for transcriptionally active genes under metabolically    relevant environmental conditions, comprises: growing said plurality    of microbial species in intimate association with a target plant    under field conditions.-   39. The guided microbial remodeling method according to any one of    the embodiments 1-7, wherein assaying the plurality of microbial    species for colonization metrics and transcriptionally active genes    under metabolically relevant environmental conditions, comprises:    growing said plurality of microbial species in intimate association    with a target plant under field conditions.-   40. The guided microbial remodeling method according to any one of    the embodiments 1-7, wherein step c) comprises assaying the    plurality of microbial species for ability to perform a    plant-beneficial function of interest under greenhouse or lab based    conditions.-   41. The guided microbial remodeling method according to any one of    the embodiments 1-7, wherein step c) comprises assaying the    plurality of microbial species for nitrogen fixation activity.-   42. The guided microbial remodeling method according to any one of    the embodiments 1-7, wherein step c) comprises assaying the    plurality of microbial species for nitrogen fixation activity in an    acetylene reduction assay or ammonium excretion assay.-   43. The guided microbial remodeling method according to any one of    the embodiments 1-42, wherein the transformation plasmid of step e)    is a suicide plasmid.-   44. The guided microbial remodeling method according to any one of    the embodiments 1-43, wherein step h) comprises whole genome    sequencing.-   45. A guided microbial remodeling method for the rational    improvement of plant-associated microbes to perform plant-beneficial    functions, comprising:    -   a. providing a plurality of microbial species;    -   b. assaying the plurality of microbial species for: colonization        metrics, transcriptionally active genes under metabolically        relevant environmental conditions, and ability to perform a        plant-beneficial function of interest;    -   c. selecting a candidate microbial species from the plurality of        assayed microbial species;    -   d. introducing one or more targeted non-intergeneric genetic        variations into the candidate microbial species at a target        genomic locus in one or more genomic pathways, or sets of genes,        that are associated with a plant-beneficial function of        interest, said non-intergeneric genetic variations selected from        the group consisting of: full gene deletions, partial gene        deletions, promoter insertions, single base pair changes, and        combinations thereof;    -   e. sequencing the genome of the candidate microbial species and        confirming integration of the non-intergeneric genetic variation        at the target genomic locus and absence of any transgenic        genetic sequence; and    -   f. repeating steps d)-e) one or more times, until a candidate        microbial species has acquired an improved ability to perform        the plant-beneficial function of interest.-   46. A guided microbial remodeling method for the rational    improvement of plant-associated microbes to perform plant-beneficial    functions, comprising:    -   a. providing a plurality of microbial species;    -   b. assaying the plurality of microbial species for: colonization        metrics, transcriptionally active genes under metabolically        relevant environmental conditions, and ability to perform a        plant-beneficial function of interest;    -   c. selecting a candidate microbial species from the plurality of        assayed microbial species;    -   d. introducing two or more targeted non-intergeneric genetic        variations into the candidate microbial species at two or more        target genomic loci, in one or more genomic pathways, or sets of        genes, that are associated with a plant-beneficial function of        interest, said non-intergeneric genetic variations selected from        the group consisting of: full gene deletions, partial gene        deletions, promoter insertions, single base pair changes, and        combinations thereof; and    -   e. sequencing the genome of the candidate microbial species and        confirming introduction of the non-intergeneric genetic        variations at the target genomic loci and absence of any        transgenic genetic sequence.-   47. A computationally guided microbial remodeling method for the    rational improvement of plant-associated microbes to perform    plant-beneficial functions, comprising:    -   a. accessing a plurality of microbial whole genome sequences;    -   b. identifying a plurality of regulatory gene sequences that        actively regulate the transcription of a gene under a        metabolically relevant environmental condition;    -   c. identifying a plurality of genes associated with a        plant-beneficial function;    -   d. selecting a regulatory gene sequence and a gene associated        with a plant-beneficial function from said pluralities; wherein        steps a)-d) occur in silico; and    -   e. manufacturing, in vivo, a remodeled microbial cell that has        the selected regulatory gene sequence operably linked to the        selected gene associated with a plant-beneficial function,        thereby improving the expression of the gene associated with a        plant-beneficial function.-   48. A computationally guided microbial remodeling system for the    rational improvement of plant-associated microbes to perform    plant-beneficial functions, comprising:    -   a. one or more processors; and    -   b. one or more memories operatively coupled to the one or more        processors and having instructions stored thereon, that when        executed by the one or more processors, cause the system to:        -   i. access a plurality of microbial whole genome sequences;        -   ii. identify a plurality of regulatory gene sequences that            actively regulate the transcription of a gene under a            metabolically relevant environmental condition;        -   iii. identify a plurality of genes associated with a            plant-beneficial function; and        -   iv. select a regulatory gene sequence and a gene associated            with a plant-beneficial function from said pluralities.-   49. A computationally guided microbial remodeling method for the    rational improvement of plant-associated microbes to perform    plant-beneficial functions, comprising:    -   a. activating a computer system having: one or more processors        and one or more memories operatively coupled to the one or more        processors and having instructions stored thereon, thereby        causing the one or more processors to execute the instructions,        and cause the system to:        -   i. access a plurality of microbial whole genome sequences;        -   ii. identify a plurality of regulatory gene sequences that            actively regulate the transcription of a gene under a            metabolically relevant environmental condition;        -   iii. identify a plurality of genes associated with a            plant-beneficial function;        -   iv. select a regulatory gene sequence and a gene associated            with a plant-beneficial function from said pluralities; and    -   b. manufacturing, in vivo, a remodeled microbial cell that has        the selected regulatory gene sequence operably linked to the        selected gene associated with a plant-beneficial function,        thereby improving the expression of the gene associated with a        plant-beneficial function.-   50. A guided microbial remodeling method for the rational    improvement of plant-associated microbes to perform plant-beneficial    functions, comprising:    -   a. providing a plurality of microbial species that are        associated with a target plant of interest;    -   b. assaying the plurality of microbial species for: colonization        metrics, and ability to perform a plant-beneficial function of        interest;    -   c. selecting a candidate microbial species from the plurality of        assayed microbial species;    -   d. introducing one or more targeted non-intergeneric genetic        variations into the candidate microbial species;    -   e. confirming integration of the non-intergeneric genetic        variation at the target genomic locus and absence of any        transgenetic sequence; and    -   f. repeating steps d) and e) one or more times, until the        candidate microbial species has acquired an improved ability to        perform the plant-beneficial function of interest.-   51. The guided microbial remodeling method according to embodiment    50, wherein the step b) comprises assaying the plurality of    microbial species for transcriptionally active genes under    metabolically relevant environmental conditions.-   52. The guided microbial remodeling method according to embodiment    50, comprising a step of sequencing the genomes of the plurality of    microbial species obtained in step a) and characterizing one or more    genomic pathways, or sets of genes, that are associated with a    plant-beneficial function of interest.-   53. The guided microbial remodeling method according to embodiment    50, wherein the step d) of introducing one or more targeted    non-intergeneric genetic variations into the candidate microbial    species comprises:    -   a. transforming the candidate microbial species with a        transformation plasmid comprising:        -   i. a selection marker,        -   ii. a counterselection marker,        -   iii. a DNA fragment comprising: a non-intergeneric genetic            variation to be introduced into the candidate microbial            species at a target genomic locus in one or more genomic            pathways, or sets of genes, that are associated with a            plant-beneficial function of interest, and homology arms to            the target genomic locus flanking the non-intergeneric            genetic variation, and        -   iv. plasmid backbone;    -   b. selecting for a candidate microbial species that has        undergone an initial homologous recombination and has the        non-intergeneric genetic variation integrated into the target        genomic locus based on the presence of the selection marker in        the genome; and    -   c. selecting for a candidate microbial species that has the        non-intergeneric genetic variation integrated into the target        genomic locus, but has undergone an additional homologous        recombination that loops-out the plasmid backbone, based on the        absence of the counterselection marker.-   54. The guided microbial remodeling method according to embodiment    50, wherein the step e) of confirming integration of the    non-intergeneric genetic variation at the target genomic locus and    absence of any transgenetic sequence comprises sequencing the genome    of the transformed candidate microbial species.-   55. The guided microbial remodeling method according to embodiment    50, wherein the step e) comprises confirming absence of any    transgenetic sequence from the transformation plasmid.-   56. The guided microbial remodeling method according to embodiment    50, wherein the plant-beneficial function of interest is nitrogen    fixation.-   57. The guided microbial remodeling method according to embodiment    50, wherein the plant-beneficial function of interest is phosphate    solubilization.-   58. The guided microbial remodeling method according to embodiment    50, wherein the plant-beneficial function of interest is microbial    colonization.-   59. The guided microbial remodeling method according to embodiment    52, wherein the step of sequencing comprises whole genome    sequencing.-   60. The guided microbial remodeling method according to embodiment    52, comprising characterizing the nitrogen fixation pathway.-   61. The guided microbial remodeling method according to embodiment    52, comprising characterizing a set of genes selected from the group    consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding    glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide    encoding glutaminase, glnD, glnE, nifJ nifH, nifD, nifK, nifY, nifE,    nifty, nifU, nifS, nif, nifW, nifZ, nifM, nifF, nifB, nifA, a gene    associated with biosynthesis of a nitrogenase enzyme, and    combinations thereof.-   62. The guided microbial remodeling method according to embodiment    52, comprising characterizing one or more genes involved in a    pathway selected from the group consisting of: exopolysaccharide    production, endo-polygalaturonase production, trehalose production,    and glutamine conversion.-   63. The guided microbial remodeling method according to embodiment    52, comprising characterizing one or more genes selected from the    group consisting of: bcsii, bcsiii, yjbE, fhaB, pehA, otsB, treZ,    glsA2, and combinations thereof-   64. The guided microbial remodeling method according to embodiment    52, comprising characterizing one or more genes selected from the    group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding    glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide    encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY,    nifE, nifty, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifA, a    gene associated with biosynthesis of a nitrogenase enzyme, bcsii,    bcsiii, yjbE, fhaB, pehA, otsB, treZ, glsA2, and combinations    thereof-   65. The guided microbial remodeling method according to embodiment    50, wherein step b) comprises assaying the plurality of microbial    species for colonization metrics under greenhouse or lab based    conditions.-   66. The guided microbial remodeling method according to embodiment    50, wherein step b) comprises assaying the plurality of microbial    species for colonization metrics under field conditions.-   67. The guided microbial remodeling method according to embodiment    50, wherein step b) comprises assaying the plurality of microbial    species for colonization metrics under greenhouse or lab based    conditions, and also under field conditions.-   68. The guided microbial remodeling method according to embodiment    50, wherein a colonization metric assayed in step b) comprises at    least one of the following: spatial colonization patterns, temporal    colonization dynamics, density of colonization, or combinations    thereof-   69. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs under greenhouse or lab based    conditions.-   70. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs under greenhouse or lab based    conditions and comprises measuring the transcriptomic profile of the    microbial species.-   71. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs under greenhouse or lab based    conditions and comprises measuring the transcriptomic activity of    genes associated with the microbial species ability to perform a    plant-beneficial function of interest.-   72. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs under greenhouse or lab based    conditions and comprises measuring the transcriptomic activity of    regulatory gene sequences.-   73. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs under greenhouse or lab based    conditions and comprises measuring the transcriptomic activity of    promoter sequences.-   74. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs under greenhouse or lab based    conditions and comprises measuring the transcriptomic activity of    promoter sequences in the presence of exogenous nitrogen.-   75. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs under greenhouse or lab based    conditions and comprises measuring the transcriptomic activity of    promoter sequences in the presence of exogenous nitrogen, wherein    said transcriptomic activity of the promoter sequences is measured    by quantifying the expression of a regulated gene.-   76. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs under field conditions.-   77. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs under field conditions and comprises    measuring the transcriptomic profile of the microbial species.-   78. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs under field conditions and comprises    measuring the transcriptomic activity of genes associated with the    microbial species ability to perform a plant-beneficial function of    interest.-   79. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs under field conditions and comprises    measuring the transcriptomic activity of regulatory gene sequences.-   80. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs under field conditions and comprises    measuring the transcriptomic activity of promoter sequences.-   81. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs under field conditions and comprises    measuring the transcriptomic activity of promoter sequences in the    presence of exogenous nitrogen.-   82. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs under field conditions and comprises    measuring the transcriptomic activity of promoter sequences in the    presence of exogenous nitrogen, wherein said transcriptomic activity    of the promoter sequences is measured by quantifying the expression    of a regulated gene.-   83. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs in vitro.-   84. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs in vitro and comprises measuring the    transcriptomic profile of the microbial species.-   85. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs in vitro and comprises measuring the    transcriptomic activity of genes associated with the microbial    species ability to perform a plant-beneficial function of interest.-   86. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs in vitro and comprises measuring the    transcriptomic activity of regulatory gene sequences.-   87. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs in vitro and comprises measuring the    transcriptomic activity of promoter sequences.-   88. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs in vitro and comprises measuring the    transcriptomic activity of promoter sequences in N-depleted and    N-replete conditions.-   89. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions occurs in vitro and comprises measuring the    transcriptomic activity of promoter sequences in N-depleted and    N-replete conditions, wherein said transcriptomic activity of the    promoter sequences is measured by quantifying the expression of a    regulated gene.-   90. The guided microbial remodeling method according to embodiment    50, wherein assaying the plurality of microbial species for    colonization metrics, comprises: growing said plurality of microbial    species in intimate association with a target plant.-   91. The guided microbial remodeling method according to embodiment    50, wherein assaying the plurality of microbial species for    colonization metrics, comprises: growing said plurality of microbial    species in intimate association with a target plant under greenhouse    or lab based conditions.-   92. The guided microbial remodeling method according to embodiment    50, wherein assaying the plurality of microbial species for    colonization metrics, comprises: growing said plurality of microbial    species in intimate association with a target plant under field    conditions.-   93. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions, comprises: growing said plurality of    microbial species in intimate association with a target plant.-   94. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions, comprises: growing said plurality of    microbial species in intimate association with a target plant under    greenhouse or lab based conditions.-   95. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    transcriptionally active genes under metabolically relevant    environmental conditions, comprises: growing said plurality of    microbial species in intimate association with a target plant under    field conditions.-   96. The guided microbial remodeling method according to embodiment    51, wherein assaying the plurality of microbial species for    colonization metrics and transcriptionally active genes under    metabolically relevant environmental conditions, comprises: growing    said plurality of microbial species in intimate association with a    target plant under field conditions.-   97. The guided microbial remodeling method according to embodiment    50, wherein step b) comprises assaying the plurality of microbial    species for ability to perform a plant-beneficial function of    interest under greenhouse or lab based conditions.-   98. The guided microbial remodeling method according to embodiment    50, wherein step b) comprises assaying the plurality of microbial    species for nitrogen fixation activity.-   99. The guided microbial remodeling method according to embodiment    50, wherein step b) comprises assaying the plurality of microbial    species for nitrogen fixation activity in an acetylene reduction    assay or ammonium excretion assay.-   100. The guided microbial remodeling method according to embodiment    50, wherein the transformation plasmid is a suicide plasmid.-   101. The guided microbial remodeling method according to embodiment    54, wherein the sequencing comprises whole genome sequencing.-   102. A guided microbial remodeling method for the rational    improvement of plant-associated microbes to perform plant-beneficial    functions, comprising:    -   a. providing a plurality of microbial species;    -   b. assaying the plurality of microbial species for: colonization        metrics, and ability to perform a plant-beneficial function of        interest;    -   c. selecting a candidate microbial species from the plurality of        assayed microbial species;    -   d. introducing one or more targeted non-intergeneric genetic        variations into the candidate microbial species at a target        genomic locus in one or more genomic pathways, or sets of genes,        that are associated with a plant-beneficial function of        interest;    -   e. confirming integration of the non-intergeneric genetic        variation at the target genomic locus and absence of any        transgenic genetic sequence; and    -   f. repeating steps d)-e) one or more times, until a candidate        microbial species has acquired an improved ability to perform        the plant-beneficial function of interest.-   103. The guided microbial remodeling method according to embodiment    102, wherein the step b) comprises assaying transcriptionally active    genes under metabolically relevant environmental conditions.-   104. The guided microbial remodeling method according to embodiment    102, wherein in step d), said non-intergeneric genetic variations    are selected from the group consisting of: full gene deletions,    partial gene deletions, promoter insertions, single base pair    changes, and combinations thereof.-   105. The guided microbial remodeling method according to embodiment    102, wherein step e) comprises sequencing the genome of the    candidate microbial species.-   106. A guided microbial remodeling method for the rational    improvement of plant-associated microbes to perform plant-beneficial    functions, comprising:    -   a. providing a plurality of microbial species;    -   b. assaying the plurality of microbial species for: colonization        metrics, and ability to perform a plant-beneficial function of        interest;    -   c. selecting a candidate microbial species from the plurality of        assayed microbial species;    -   d. introducing two or more targeted non-intergeneric genetic        variations into the candidate microbial species at two or more        target genomic loci, in one or more genomic pathways, or sets of        genes, that are associated with a plant-beneficial function of        interest; and    -   e. confirming introduction of the non-intergeneric genetic        variations at the target genomic loci and absence of any        transgenic genetic sequence.-   107. The guided microbial remodeling method according to embodiment    106, wherein step b) comprises assaying transcriptionally active    genes under metabolically relevant environmental conditions.-   108. The guided microbial remodeling method according to embodiment    106, wherein in step d), said non-intergeneric genetic variations    are selected from the group consisting of: full gene deletions,    partial gene deletions, promoter insertions, single base pair    changes, and combinations thereof.-   109. The guided microbial remodeling method according to embodiment    106, wherein step e) comprises sequencing the genome of the    candidate microbial species.

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosure. It should beunderstood that various alternatives to the embodiments of thedisclosure described herein may be employed in practicing thedisclosure. It is intended that the following Claims define the scope ofthe disclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes. However, mention of any reference,article, publication, patent, patent publication, and patent applicationcited herein is not, and should not be taken as, an acknowledgment orany form of suggestion that they constitute valid prior art or form partof the common general knowledge in any country in the world. Further,U.S. Pat. No. 9,975,817, issued on May 22, 2018, and entitled: Methodsand Compositions for Improving Plant Traits, is hereby incorporated byreference. Further, PCT/US2018/013671, filed Jan. 12, 2018, andentitled: Methods and Compositions for Improving Plant Traits, is herebyincorporated by reference.

1. A guided microbial remodeling method for the rational improvement ofplant-associated microbes to perform plant-beneficial functions,comprising: a. providing a plurality of microbial species that areassociated with a target plant of interest; b. assaying the plurality ofmicrobial species for: colonization metrics, and ability to perform aplant-beneficial function of interest; c. selecting a candidatemicrobial species from the plurality of assayed microbial species; d.introducing one or more targeted non-intergeneric genetic variationsinto the candidate microbial species; e. confirming integration of thenon-intergeneric genetic variation at the target genomic locus andabsence of any transgenetic sequence; and f. repeating steps d) and e)one or more times, until the candidate microbial species has acquired animproved ability to perform the plant-beneficial function of interest.2. The guided microbial remodeling method according to claim 1, whereinthe step b) comprises assaying the plurality of microbial species fortranscriptionally active genes under metabolically relevantenvironmental conditions.
 3. The guided microbial remodeling methodaccording to claim 1, comprising a step of sequencing the genomes of theplurality of microbial species obtained in step a) and characterizingone or more genomic pathways, or sets of genes, that are associated witha plant-beneficial function of interest.
 4. The guided microbialremodeling method according to claim 1, wherein the step d) ofintroducing one or more targeted non-intergeneric genetic variationsinto the candidate microbial species comprises: a. transforming thecandidate microbial species with a transformation plasmid comprising (i)a selection marker, (ii) a counterselection marker, (iii) a DNA fragmentcomprising: a non-intergeneric genetic variation to be introduced intothe candidate microbial species at a target genomic locus in one or moregenomic pathways, or sets of genes, that are associated with aplant-beneficial function of interest, and homology arms to the targetgenomic locus flanking the non-intergeneric genetic variation, and (iv)plasmid backbone; b. selecting for a candidate microbial species thathas undergone an initial homologous recombination and has thenon-intergeneric genetic variation integrated into the target genomiclocus based on the presence of the selection marker in the genome; andc. selecting for a candidate microbial species that has thenon-intergeneric genetic variation integrated into the target genomiclocus, but has undergone an additional homologous recombination thatloops-out the plasmid backbone, based on the absence of thecounterselection marker.
 5. The guided microbial remodeling methodaccording to claim 1, wherein the step e) of confirming integration ofthe non-intergeneric genetic variation at the target genomic locus andabsence of any transgenetic sequence comprises sequencing the genome ofthe transformed candidate microbial species.
 6. (canceled)
 7. The guidedmicrobial remodeling method according to claim 1, wherein theplant-beneficial function of interest is nitrogen fixation, phosphatesolubilizatoin, or microbial colonization.
 8. (canceled)
 9. (canceled)10. The guided microbial remodeling method according to claim 3, whereinthe step of sequencing comprises whole genome sequencing.
 11. The guidedmicrobial remodeling method according to claim 3, comprisingcharacterizing the nitrogen fixation pathway.
 12. (canceled)
 13. Theguided microbial remodeling method according to claim 3, comprisingcharacterizing one or more genes involved in a pathway selected from thegroup consisting of: exopolysaccharide production, endo-polygalaturonaseproduction, trehalose production, and glutamine conversion. 14.(canceled)
 15. The guided microbial remodeling method according to claim3, comprising characterizing one or more genes selected from the groupconsisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutaminesynthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encodingglutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU,nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, a gene associated withbiosynthesis of a nitrogenase enzyme, bcsii, bcsiii, yjbE, fhaB, pehA,otsB, treZ, glsA2, and combinations thereof.
 16. (canceled) 17.(canceled)
 18. The guided microbial remodeling method according to claim1, wherein step b) comprises assaying the plurality of microbial speciesfor colonization metrics under greenhouse conditions, lab basedconditions, and/or under field conditions.
 19. The guided microbialremodeling method according to claim 1, wherein a colonization metricassayed in step b) comprises at least one of the following: spatialcolonization patterns, temporal colonization dynamics, density ofcolonization, or combinations thereof.
 20. (canceled)
 21. The guidedmicrobial remodeling method according to claim 2, wherein assaying theplurality of microbial species for transcriptionally active genes undermetabolically relevant environmental conditions occurs in vitro, undergreenhouse, or lab based, or field conditions and comprises measuringthe transcriptomic profile of the microbial species.
 22. The guidedmicrobial remodeling method according to claim 2, wherein assaying theplurality of microbial species for transcriptionally active genes undermetabolically relevant environmental conditions occurs in vitro, undergreenhouse, or lab based, or field conditions and comprises measuringthe transcriptomic activity of genes associated with the microbialspecies ability to perform a plant-beneficial function of interest. 23.The guided microbial remodeling method according to claim 2, whereinassaying the plurality of microbial species for transcriptionally activegenes under metabolically relevant environmental conditions occurs undergreenhouse or lab based, or field conditions and comprises at least oneof: measuring the transcriptomic activity of regulatory gene sequences,measuring the transcriptomic activity of promotor sequences; measuringthe transcriptomic activity of promotor sequences in the presence ofexogenous nitrogen, and measuring the transcriptomic activity ofpromoter sequences in the presence of exogenous nitrogen, wherein saidtranscriptomic activity of the promoter sequences is measured byquantifying the expression of a regulated gene. 24.-36. (canceled) 37.The guided microbial remodeling method according to claim 2, whereinassaying the plurality of microbial species for transcriptionally activegenes under metabolically relevant environmental conditions occurs invitro and comprises at least one of: measuring the transcriptomicactivity of regulatory gene sequences, measuring the transcriptomicactivity of promotor sequences; measuring the transcriptomic activity ofpromotor sequences in N-depleted and N-replete conditions, and measuringthe transcriptomic activity of promoter sequences in N-depleted andN-replete conditions, wherein said transcriptomic activity of thepromoter sequences is measured by quantifying the expression of aregulated gene. 38.-41. (canceled)
 42. The guided microbial remodelingmethod according to claim 1, wherein assaying the plurality of microbialspecies for colonization metrics, comprises: growing said plurality ofmicrobial species in intimate association with a target plant undergreenhouse conditions, or lab based conditions, and/or field conditions.43. (canceled)
 44. (canceled)
 45. The guided microbial remodeling methodaccording to claim 2, wherein assaying the plurality of microbialspecies for transcriptionally active genes under metabolically relevantenvironmental conditions, comprises: growing said plurality of microbialspecies in intimate association with a target plant under greenhouseconditions, lab based conditions, or field conditions.
 46. (canceled)47. (canceled)
 48. The guided microbial remodeling method according toclaim 1, wherein step b) comprises assaying the plurality of microbialspecies for ability to perform a plant-beneficial function of interestunder greenhouse or lab based conditions.
 49. The guided microbialremodeling method according to claim 1, wherein step b) comprisesassaying the plurality of microbial species for nitrogen fixationactivity.
 50. The guided microbial remodeling method according to claim1, wherein step b) comprises assaying the plurality of microbial speciesfor nitrogen fixation activity in an acetylene reduction assay orammonium excretion assay.
 51. (canceled)
 52. The guided microbialremodeling method according to claim 5, wherein the sequencing compriseswhole genome sequencing.
 53. (canceled)
 54. (canceled)
 55. The guidedmicrobial remodeling method according to claim 1, wherein in step d),said non-intergeneric genetic variations are selected from the groupconsisting of: full gene deletions, partial gene deletions, promoterinsertions, single base pair changes, and combinations thereof. 56.(canceled)
 57. (canceled)
 58. (canceled)
 59. (canceled)
 60. (canceled)61. A computationally guided microbial remodeling method for therational improvement of plant-associated microbes to performplant-beneficial functions, comprising: a. accessing a plurality ofmicrobial whole genome sequences; b. identifying a plurality ofregulatory gene sequences that actively regulate the transcription of agene under a metabolically relevant environmental condition; c.identifying a plurality of genes associated with a plant-beneficialfunction; d. selecting a regulatory gene sequence and a gene associatedwith a plant-beneficial function from said pluralities; wherein stepsa)-d) occur in silico; and e. manufacturing, in vivo, a remodeledmicrobial cell that has the selected regulatory gene sequence operablylinked to the selected gene associated with a plant-beneficial function,thereby improving the expression of the gene associated with aplant-beneficial function.
 62. A computationally guided microbialremodeling system for the rational improvement of plant-associatedmicrobes to perform plant-beneficial functions, comprising: a. one ormore processors; and b. one or more memories operatively coupled to theone or more processors and having instructions stored thereon, that whenexecuted by the one or more processors, cause the system to: i. access aplurality of microbial whole genome sequences; ii. identify a pluralityof regulatory gene sequences that actively regulate the transcription ofa gene under a metabolically relevant environmental condition; iii.identify a plurality of genes associated with a plant-beneficialfunction; and iv. select a regulatory gene sequence and a geneassociated with a plant-beneficial function from said pluralities.
 63. Acomputationally guided microbial remodeling method for the rationalimprovement of plant-associated microbes to perform plant-beneficialfunctions, comprising: a. activating a computer system having: one ormore processors and one or more memories operatively coupled to the oneor more processors and having instructions stored thereon, therebycausing the one or more processors to execute the instructions, andcause the system to: i. access a plurality of microbial whole genomesequences; ii. identify a plurality of regulatory gene sequences thatactively regulate the transcription of a gene under a metabolicallyrelevant environmental condition; iii. identify a plurality of genesassociated with a plant-beneficial function; iv. select a regulatorygene sequence and a gene associated with a plant-beneficial functionfrom said pluralities; and b. manufacturing, in vivo, a remodeledmicrobial cell that has the selected regulatory gene sequence operablylinked to the selected gene associated with a plant-beneficial function,thereby improving the expression of the gene associated with aplant-beneficial function.